Type III secretion system effector proteins are mechanically labile

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔGunfold0) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published ΔGunfold0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (F_unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx^‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (F_unfold > 80 pN) and brittle (Δx^‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Type III secretion systems (T3SS) are large nanomachines utilized by both pathogenic and symbiotic bacteria to inject effector proteins directly into the cytoplasm of host cells (1–3). Once delivered, effector proteins facilitate host cell colonization through a variety of mechanisms (4–7), including down-regulation of the host immune response (8) and rearrangement of the cytoskeleton (9, 10). The T3SS apparatus, known as the injectisome, is a syringe-like structure with a hollow needle that spans the inner and outer bacterial membranes, the extracellular space, and the host membrane, enabling proteins to pass directly from bacteria to host cells (Fig. 1A) (2). Specialized bacterial chaperones often bind the N-terminal 50 to 100 amino acids (aa) of the effector proteins, known as the chaperone binding domain, and help maintain the effector N-terminal domain in an extended conformation. C-terminal to the chaperone binding domain, effector proteins contain one or more globular domains, which adopt their folded conformations even when in complex with their cognate chaperone (4, 11, 12). The effector proteins, or their chaperone complexes, are recognized by the base of the injectisome prior to secretion (13). At its narrowest point, the injectisome needle’s inner diameter is less than 2 nm (14–16). As a result, effector proteins must be mostly unfolded to be secreted (17–20). Secretion is thus thought to proceed by a “threading-the-needle mechanism,” where the N-terminal extended domain is released from the chaperone and fed to the injectisome, followed by unfolding of the C-terminal effector domain (21).Open in a separate windowFig. 1.Thermodynamic stability of T3SS effector proteins SptP_CD and SopE2_CD. (A) Schematic depiction of protein transport through the T3SS showing effector proteins, which are at least partially folded in the bacterial cytoplasm. Such effector proteins interact with an associated unfoldase to passage through the T3SS, which has an inner channel with a diameter <2 nm. Once inside the host cytoplasm, effector proteins refold to carry out their function. (B) Crystal structures of SptP_CD (Protein Data Bank [PDB] ID code 1G4U) and SopE2_CD (PDB ID code 1R9K). (C) Ellipticity from CD at λ = 222 nm plotted as a function of urea concentrations for SptP_CD (orange) and SopE2_CD (green). A fit of the data with Eq. 1 yielded the free energy of unfolding ΔGunfold0 for SptP_CD (6.9 ± 0.2 kcal/mol [mean ± fit error]) and SopE2_CD (6.0 ± 0.2 kcal/mol [mean ± fit error]). Data points are the result of at least three independent measurements. Error bars represent SD.Before proteins are secreted through the T3SS, they interact with a hexameric ATPase at the base of the T3SS that is capable of mediating chaperone release from effector proteins and effector-protein unfolding (15, 22). Indeed, most in vivo unfolding is catalyzed by unfoldases that work from one end of the substrate protein in stark contrast to the global effects of temperature, pH, or chemical denaturants. The most common examples of targeted protein unfolding are catalyzed by ATPases of the AAA(+) family that mechanically unfold their substrates (23, 24). For example, the AAA(+) ATPase ClpX forms a ring-shaped hexamer that mechanically pulls its substrates through its narrow central pore to unfold them (25). These are powerful unfoldases that can unfold even tightly packed proteins such as GFP, ubiquitin, and dihydrofolate reductase (DHFR) (23, 24, 26, 27). However, the T3SS ATPase does not belong to the AAA(+) family of ATPases. Instead, it is structurally similar to the catalytic β-subunit of the F₁F₀ ATP synthase, a rotary motor that normally couples proton gradient dissipation to ATP synthesis but can also run in reverse and hydrolyze ATP to do work (15, 28–30). The T3SS ATPase is not as powerful an unfoldase as the AAA(+) family, as fusions of effector proteins with GFP, ubiquitin, or DHFR stall in the injectisome and are poorly secreted (20, 22, 31, 32). These observations have led to the current model that T3SS effector proteins have low thermodynamic stability to facilitate their secretion (22, 31–33).While thermodynamic stability is the most common metric of protein stability, mechanical stability is a distinct metric that quantifies how easily a protein unfolds under force (F_unfold). Mechanical stability is typically measured by pulling across the N and C termini of single molecules via force spectroscopy using optical tweezers (34, 35) or an atomic force microscope (AFM) (36). Early force spectroscopy studies showed that thermodynamic stability does not correlate with mechanical stability (37–41). For example, titin’s I28 domain requires ∼20% more force to unfold than titin’s I27 domain [I85 and I91, respectively, in the new nomenclature (42)], despite I27 having approximately twofold higher thermodynamic stability (43). Importantly, AFM studies have shown that GFP (44), ubiquitin (45), and DHFR (46) are mechanically robust, requiring high forces to unfold despite their typical thermodynamic stabilities. These three proteins each stall the T3SS; thus, mechanical stability may be the physical determinant to proteins being secreted by the T3SS, rather than thermodynamic stability.Here, we determine the thermodynamic and mechanical stabilities of SptP and SopE2, two effector proteins from Salmonella enterica. These effectors are ideal candidates for this study as they have known crystal structures (10, 47), have characterized in vivo secretion kinetics (48), and represent effector proteins of different size and structure (Fig. 1B). We show that the catalytic domains of SptP and SopE2 have unremarkable thermodynamic stabilities, similar to many other previously characterized proteins, including GFP, ubiquitin, and DHFR. Conversely, our AFM-based force spectroscopy measurements demonstrate that SptP and SopE2 are among the most mechanically labile proteins studied to date by AFM. These two T3SS effector proteins are therefore mechanically labile while being thermodynamically stable, supporting the hypothesis that it is mechanical stability, not thermodynamic stability, that predicts efficient protein secretion by the T3SS.     

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔG for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val²¹⁷ and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.

Membrane proteins play a critical role in metabolism, transport, and signaling. Membrane proteins are also of intense biomedical interest, as they are the target for ∼50% of approved drugs (1). X-ray crystallography (2) and, more recently, cryo-electron microscopy (3–5) are yielding an accelerating number of high-resolution structures. Yet, predicting the folding and dynamics of membrane proteins remains an unmet need, in part, because it remains difficult to characterize the energetics that drive and stabilize a membrane protein’s folded structure (6–9). Membrane protein energetics are characterized in terms of the unfolding free energy (ΔG) and the change in that free energy upon introduction of a single amino acid point mutation (ΔΔG). Ideally, ΔG is interpretable as the sum of stabilizing interactions in the protein (e.g., van der Waals, hydrogen bonding), and ΔΔG isolates the energetic contribution of a single amino acid side chain to that stability. However, such an interpretation is only possible if the measurement technique reports the underlying molecular energetics with fidelity. For example, a measurement that is not made in the native bilayer may fail to accurately report stabilizing amino acid–lipid interactions. Here, we establish a single-molecule platform for measuring ΔΔG in bacteriorhodopsin (bR), a model membrane protein. The method is based upon mechanical unfolding of individual molecules in their native lipid bilayer (Fig. 1A), avoiding the nonnative detergent environment and the perturbative chemical denaturant used in traditional ensemble biochemical assays (Fig. 1B).Open in a separate windowFig. 1.Membrane protein energetics assays. (A) Illustration of the single-molecule mechanical unfolding assay where bR in its native bilayer is deposited onto a mica surface. An AFM cantilever then applies tension to the C-terminal tail, causing an eight-aa region (cyan) to reversibly unfold to a taut unfolded state (Inset). Unfolding is measured by a change in the polypeptide extension x. (B) Illustration of the traditional chemical denaturation assay showing reconstituted bR in a mixed micelle. Application of SDS then leads to a denatured state that retains ∼60% of its secondary α-helical structure (cylinders).Chemical denaturation of at least 85 bR point mutants (10–18) helps form the foundation for the current understanding of membrane protein energetics (8, 19) despite several key limitations. These measurements typically begin with reconstituted bR folded in a nonnative mixed micelle of phospholipid and detergent. Introduction of sodium dodecyl sulfate (SDS) then reversibly denatures the protein in a single step, disrupting the tertiary structure but leaving a significant fraction of secondary structure intact [∼60% as measured by circular dichroism (20)] (Fig. 1B). Analysis of the denaturant concentration dependence yields ΔG and, when repeated for point mutants, ΔΔG (21). However, this widely applied technique has four underlying limitations that can bias measured values. First, the bR is solvated in a nonnative mixed micelle, causing the free energy of the folded state to lack the energetic contributions from native protein–lipid interactions (22). Second, the measurements involve extrapolation from high denaturant concentration to zero, which assumes a linearity that steric-trapping experiments have called into question (23). Third, the free energy of the denatured state includes contributions from the ∼60% residual secondary structure (20, 24) and from nonspecific interactions with the denaturant (25). Fourth, the vast majority of membrane proteins—including all G-protein–coupled receptors—are not amenable to these measurements because there is no currently known condition under which their chemical denaturation is reversible (26).Force-induced mechanical unfolding represents an alternative means for measuring ΔΔG without the underlying limitations of chemical denaturation assays. In these atomic force microscopy (AFM) studies, the protein is initially folded in a lipid bilayer—native purple membrane in the case of bR—providing sensitivity to protein–lipid and quaternary interactions (27, 28). The cantilever of an AFM then pulls on one end of an individual membrane protein, causing it to unfold under force (F) to a stretched state (i.e., a highly extended polypeptide chain). This taut, unfolded state emerges from the bilayer into the surrounding buffer and thus contains no energetic contributions from residual secondary structure (29) or detergent interactions (Fig. 1A). As a result, both the folded and unfolded states are thermodynamically well defined, in contrast to the “unfolded” state induced by SDS denaturation (Fig. 1B). When probed with sufficient sensitivity, the unfolding process is shown to occur via a multitude of discrete states (30), each corresponding to a metastable unfolding intermediate. Reversible transitions between these states—some separated by the folding of as few as two to three amino acids (aa) (30, 31)—allow for local equilibrium energetic measurements of proteins that do not reversibly refold on larger scales.Many studies have reported AFM-based mechanical unfolding of bR (27, 30–40), including some reporting unfolding free energies (41–43); however, most prior studies lacked the ability to observe reversible transitions in the segments of bR that unfold first. In particular, assays that rely on nonspecific tip-sample attachment suffer from surface adhesion that prevents precise characterization of the first two of bR’s seven transmembrane helices to be extracted. These assays therefore cannot quantify the energetics of bR when the most native set of interactions are present. Nonspecific attachment is also not mechanically robust, preventing repeated unfolding and refolding over an extended period. Additionally, most prior AFM measurements of bR were far from equilibrium, did not account for the anomalous work of stretching the unfolded polypeptide, and had insufficient spatiotemporal resolution to observe occupancies of short-lived, closely spaced unfolding intermediates.Recent technological and methodological improvements have overcome the limitations of prior AFM-based force spectroscopy assays to allow the energetics of bR to be measured on the scale of a few aa. In particular, Yu et al. (31) showed that copper-free click chemistry can be used to form, in situ, a site-specific covalent bond between bR and an AFM tip. This chemistry allows for longer data collection and for smaller surface-contact forces, the latter reducing adhesion and enabling interpretation of the first eight aa to unfold. Additionally, Edwards et al. (44) developed focused ion beam (FIB)-modified cantilevers that provided a 100-fold improvement in time resolution and 10-fold improvement in force precision over prior studies of bR (27, 28). Recently, we combined these techniques to characterize the unfolding and refolding of the first eight aa at the C terminus of wild-type (wt) bR’s helix G, while the rest of the protein remained folded in native purple membrane. From these data, we deduced values of ΔG for these residues (∼2 kcal/mol per aa) that were consistent between three distinct analyses using both equilibrium and near-equilibrium protocols (45). Notably, this ΔG is ∼20-fold larger (on a per aa basis) than a prior chemical denaturation measurement (16). We attributed this difference in ΔG to the differing unfolded states between the two assays, with the SDS-denaturation measurement not accounting for the energy needed to disrupt the full α-helical secondary structure of bR and to solvate the unfolded protein into water (Fig. 1). Given this large difference in ΔG, it remained an open question whether consistent values of ΔΔG could be obtained between the two assays.Here, we applied these recently developed AFM techniques to measure ΔΔG of two alanine point mutants (L223A and V217A) that were previously characterized using SDS denaturation (16) and that probed different side-chain environments. Leu²²³ is oriented into the protein core of bR, whereas Val²¹⁷ is orientated outwards toward the lipid environment of purple membrane and contributes to the binding pocket of a crystallographically resolved squalene lipid (46). Ideally, ΔΔG values would agree between AFM unfolding and SDS denaturation, as both are meant to reflect the underlying contribution of the mutated side chain to the overall protein stability. Interestingly, our ΔΔG value for L223A agreed quantitatively with prior SDS-denaturation experiments despite the vastly different ΔG for the two assays. However, the AFM-based determination of ΔΔG for V217A was 2.2-fold larger than the SDS-based value, highlighting the strength of lipid–protein interactions and the importance of characterizing membrane protein energetics in the native bilayer. These measurements, therefore, constitute a practical demonstration of a widely applicable, single-molecule technique for measuring membrane protein thermodynamics that is not subject to the limitations of chemical denaturation experiments.     

A new sea-level record for the Neogene/Quaternary boundary reveals transition to a more stable East Antarctic Ice Sheet

Sea-level rise resulting from the instability of polar continental ice sheets represents a major socioeconomic hazard arising from anthropogenic warming, but the response of the largest component of Earth’s cryosphere, the East Antarctic Ice Sheet (EAIS), to global warming is poorly understood. Here we present a detailed record of North Atlantic deep-ocean temperature, global sea-level, and ice-volume change for ∼2.75 to 2.4 Ma ago, when atmospheric partial pressure of carbon dioxide (pCO₂) ranged from present-day (>400 parts per million volume, ppmv) to preindustrial (<280 ppmv) values. Our data reveal clear glacial–interglacial cycles in global ice volume and sea level largely driven by the growth and decay of ice sheets in the Northern Hemisphere. Yet, sea-level values during Marine Isotope Stage (MIS) 101 (∼2.55 Ma) also signal substantial melting of the EAIS, and peak sea levels during MIS G7 (∼2.75 Ma) and, perhaps, MIS G1 (∼2.63 Ma) are also suggestive of EAIS instability. During the succeeding glacial–interglacial cycles (MIS 100 to 95), sea levels were distinctly lower than before, strongly suggesting a link between greater stability of the EAIS and increased land-ice volumes in the Northern Hemisphere. We propose that lower sea levels driven by ice-sheet growth in the Northern Hemisphere decreased EAIS susceptibility to ocean melting. Our findings have implications for future EAIS vulnerability to a rapidly warming world.

The instability of polar continental ice sheets in a warmer future is an issue of major societal concern (1–5). Based on linear extrapolation of recent sea-level rise (2), mean global sea level could increase by 65 ± 12 cm by 2100 relative to the 2005 baseline, consistent with Intergovernmental Panel on Climate Change projections (1) of a ∼30- to 100-cm increase by 2100. Further, satellite observations (4) document substantial mass loss of both the Greenland Ice Sheet (GIS) and the West Antarctic Ice Sheet (WAIS) over the past decade—the two ice sheets that are most susceptible to global warming because of rapidly rising Arctic air temperatures (1) (GIS) and vulnerability to ocean-atmospheric warming (5, 6) (WAIS). The mass balance of the much larger EAIS and its contribution to ongoing sea-level change, however, remain poorly constrained (1).The role of atmospheric partial pressure of carbon dioxide (pCO₂) as a driver of long-term changes in ice volume and sea level over the Cenozoic Era (past ∼66 My) is widely documented (7–9) and there is compelling evidence (6, 10–12) of East Antarctic Ice Sheet (EAIS) retreat during warm intervals of the Pliocene epoch between ∼5.3 and 3.3 Ma when pCO₂ levels (13, 14) last reached values close to the present day (∼400 parts per million volume [ppmv]; Fig. 1 A and B and see SI Appendix, section S1). However, there is disagreement over EAIS behavior under pCO₂ levels (13) similar to those of preindustrial Quaternary times (<280 ppmv). A compilation of marine geochemical paleo-sea-level and pCO₂ records suggests that the EAIS was stable under these conditions (7). In contrast, while the amplitudes of change are controversial (15) (SI Appendix, section S2), sea-level reconstructions from paleoshorelines (16) and benthic geochemical data (9, 17, 18) (Fig. 2) imply EAIS melting during the Quaternary “super-interglacials” of Marine Isotope Stage (MIS) 11 (∼400 ka) and 31 (∼1.07 Ma) under relatively low pCO₂ conditions. Supporting evidence for EAIS retreat during the most recent “super-interglacial” MIS 11 comes from isotope measurements in mineral deposits recording past changes in subglacial East Antarctic waters (19), as well as records of ice-rafted debris (IRD) and detrital sediment neodymium isotopes from offshore the Wilkes Subglacial Basin (20). The latter records (20) also indicate EAIS retreat during the last interglacial MIS 5e (∼120 ka). Melting of the EAIS as inferred in the late Quaternary was likely driven by ocean–atmosphere warming around Antarctica and grounding-line retreat in response to ice–ocean interactions (19, 20).Open in a separate windowFig. 1.Neogene to Quaternary climate and sea-level evolution. (A) LR04 stack (21) for the past 5 My; arrow indicates the iNHG (∼3.6 to 2.4 Ma) and its culmination (thick-arrowed interval) (22); green line indicates the benthic δ¹⁸O level associated with MIS 101. (B) Atmospheric pCO₂ estimates of refs. 13 (blue) and 23 (purple) for the past 5 My; the late Quaternary glacial–interglacial pCO₂ range (1) is indicated as preindustrial pCO₂ band. Yellow shading in A and B highlights the study interval (∼2.75 to 2.4 Ma). (C and D) Site U1313 benthic δ¹⁸O and Mg/Ca raw data, respectively. (E) Site U1313 deep-sea temperature. (F) Site U1313 δ¹⁸O_sw-based sea level relative to present (black line); blue shading: 95% probability interval from Monte Carlo simulations (2σ); red line: threshold (11.6 msle) above which a smaller-than-present EAIS is signaled (24–26); m = marine part of EAIS, t = terrestrial part of EAIS. Glacials are highlighted in gray.Open in a separate windowFig. 2.Implication of different sea-level-δ¹⁸O_sw conversions for estimates of interglacial ice-volume loss. y axis shows lower-than-modern δ¹⁸O_sw values (∆δ¹⁸O_sw) and the x axis (log-scale) the corresponding sea-level increase for commonly used conversion factors (27–29) (0.011 [black], 0.010 [purple], and 0.008 ‰⋅m⁻¹ [red]) and those for Antarctica only (11) (0.014 ‰⋅m⁻¹) ignoring (yellow) and incorporating (brown) the impact of its marine-based ice sheets. Stars mark ∆δ¹⁸O_sw for interglacials of this study and corresponding sea-level equivalents in dependence of the conversion applied. Orange, blue, and purple diamonds show the same for MIS 31, 11 (18), and 5e (17), respectively. Vertical lines indicate the sea-level increase resulting from complete melting of the GIS (+7.3 m), WAIS (+4.3 m), and EAIS (+53.3 m) (24–26).To further investigate past EAIS response to climate forcing we studied the Neogene/Quaternary transition when mean pCO₂ (13, 23) fell from levels similar to the anthropogenically perturbed values of today into the Quaternary range, leading to progressive high-latitude cooling and the intensification of Northern Hemisphere Glaciation (21, 30–33) (iNHG; Fig. 1 A and B). Our approach is based on a simple approximation that, once estimated past global sea level exceeds 11.6 m sea-level equivalent (msle) above modern, which corresponds to the complete melting of the present-day GIS [7.3 msle (24, 25)] and the marine- and land-based WAIS [3.4 and 0.9 msle (25, 26), respectively], EAIS instability (i.e., a retreat from its present-day size) can be inferred (see SI Appendix, section S4.1 for details). We quantified sea-level and ice-volume changes for the interval ∼2.75 to 2.4 Ma (MIS G7 to 95) by measuring the oxygen-isotope composition (δ¹⁸O) and Mg/Ca ratio in well-preserved benthic foraminiferal calcite (Oridorsalis umbonatus) from Integrated Ocean Drilling Program (IODP) Site U1313 [41°0′N, 32°57′W; 3,426-m water depth (34)] in the North Atlantic Ocean (Fig. 1 C and D). Using this approach we reconstructed changes in seawater δ¹⁸O (δ¹⁸O_sw), a proxy for global sea level and continental ice volume (35). This was done by 1) calculating bottom-water temperatures (BWT) derived from Mg/Ca (36) (Fig. 1E), 2) combining Mg/Ca-derived BWTs with δ¹⁸O to determine δ¹⁸O_sw (37) (Fig. 1F), and 3) converting δ¹⁸O_sw to sea level using a relationship between changes in sea level and δ¹⁸O_sw of 0.011 ‰⋅m⁻¹ (27) (Materials and Methods and Fig. 1F). Ninety-five percent probability intervals calculated through Monte Carlo simulations for individual sea-level data points yield an average uncertainty for our sea-level estimates of ± 28 m (∼2σ [SD]) (Materials and Methods and Fig. 1F), roughly equivalent to the decay/growth of ice four times greater than the GIS. Our approach was validated by reconstructing δ¹⁸O_sw for the recent (∼0 to 7 ka) at IODP Site U1313 and for late Holocene core-top (multicorer) samples from a neighboring site (MSM58) which are indistinguishable from the observed modern-day values (see Materials and Methods and SI Appendix, section S4.2.8 for details).     

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

L-type voltage-gated Ca_V1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)–induced increase of calcium influx through Ca_V1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a Ca_V1.2 inhibitory protein, as essential for PKA regulation of Ca_V1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of Ca_V1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified Ca_V1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR–Ca_V1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Cardiac excitation–contraction coupling crucially depends on the L-type voltage-dependent Ca²⁺ channel, Ca_V1.2. Influx of extracellular Ca²⁺ via Ca_V1.2 triggers Ca²⁺ release from the sarcoplasmic reticulum via the Ca²⁺ release channel (1). Activation of the sympathetic nervous system increases heart rate, relaxation rate and contraction force. The latter is largely due to increased Ca²⁺ influx via Ca_V1.2 (2, 3). Pathological prolonged sympathetic activation progressively impairs cardiac function, causing heart failure, partly due to misregulation of Ca_V1.2 (4, 5).Cardiac Ca_V1.2 is a heterotrimer comprising the pore-forming subunit α_1C (∼240 kDa), the intracellular Ca_Vβ₂ (∼68 kDa) and the extracellular α2δ (∼170 kDa) (Fig. 1A) (6, 7). The N and C termini (NT, CT respectively) of α_1C are cytosolic and vary among Ca_V1.2 isoforms. Further, most of the cardiac α_1C protein is posttranslationally cleaved at the CT, around amino acid (a.a.) 1800, to produce the truncated ∼210-kDa α_1C protein and the ∼35-kDa cleaved distal CT (dCT); however, the full-length protein is also present (8–11).Open in a separate windowFig. 1.cAMP regulation of Ca_V1.2 is enhanced by coexpression of Rad. (A) Ca_V1.2 and Rad. α_1C and α2δ subunits are shown schematically, with structures of β_2b (38) and Rad (74). The truncation in α_1CΔ1821 was at a.a. 1,821 (red cross mark) similar to naturally truncated cardiac α_1C, ∼a.a. 1800 (9). Ca_Vβ binds to the cytosolic loop I, L1, that connects repeat domains I and II. Rad exerts inhibitory action on the channel, in part through an interaction with Ca_Vβ. (B) Rad reduces the Ba²⁺ current of Ca_V1.2-α_1CΔ1821 (α_1CΔ1821, β_2b and α2δ; 1.5 ng RNA of each subunit) in a dose-dependent manner. Pearson correlation, r = −0.82, P = 0.023. Each point represents mean ± SEM from 7 to 10 oocytes recorded during 1 d. The linear regression line was drawn for nonzero doses of Rad. (C) Rad enhances the cAMP-induced increase in I_Ba. Diary plots of the time course of change in I_Ba (normalized to initial I_Ba) are shown before and after intracellular injection of cAMP in representative cells. No Rad: Upper; with Rad: Lower. (Insets) Currents at +20 mV before (black trace) and 10 min after cAMP injection (red trace). (D) “before–after” plots of cAMP-induced changes in I_Ba in individual cells injected Rad RNA while varying Rad:β_2b RNA ratio (by weight, wt/wt). Empty symbols–before cAMP; red-filled–after cAMP. n = 3 experiments; statistics: paired t test. (E) cAMP-induced increase in I_Ba at different Rad/β_2b RNA levels (summary of data from D). Each symbol represents fold increase in I_Ba induced by cAMP injection in one cell. Here and in the following figures, box plots show 25 to 75 percentiles, whiskers show the 5/95 percentiles, and black and red horizontal lines within the boxes are the median and mean, respectively. At all Rad:β_2b RNA ratios except 1:20, the cAMP-induced increase in I_Ba was significantly greater than without Rad (Kruskal–Wallis test; H = 36.1, 6 degrees of freedom, P < 0.001). (F) Summary of cAMP effects in 10 experiments without and with Rad at 1:2 and 1:1 Rad:β_2b RNA ratios (pooled). Number of cells: within the bars. Statistics: Mann–Whitney U test; U = 19.0, P < 0.001.The sympathetic nervous system activates cardiac β-adrenergic receptors (β-AR), primarily β1-AR (which is coupled to G_s, is globally distributed in cardiomyocytes, and mediates most of the β-AR-enhancement of contraction and Ca_V1.2 activity) and β2-AR, which can couple to both G_s and G_i (12). The cascade of adrenergic modulation of Ca_V1.2 comprises agonist binding to β-ARs, activation of G_s and adenylyl cyclase, elevated intracellular cAMP levels, and activation of protein kinase A (PKA) by cAMP-induced dissociation of its catalytic subunit (PKA-CS) from the regulatory subunit. However, the final step, how PKA-CS enhances Ca_V1.2 activity, remained enigmatic. A long-standing paradigm was a direct phosphorylation by PKA-CS of α_1C and/or Ca_Vβ subunits (3, 13–16). However, numerous studies critically challenged this theory. In particular, mutated Ca_V1.2 channels in genetically engineered mice lacking putative PKA phosphorylation sites on α_1C and/or β_2b, were still up-regulated by PKA (9, 17–21) (reviewed in refs. 6 and 22).One significant obstacle in deciphering the mechanism of PKA regulation of Ca_V1.2 was a recurrent lack of success in reconstituting the regulation in heterologous systems, which proved challenging and controversial (23). Studies in heterologous cellular models, including Xenopus oocytes, demonstrated that cAMP failed to up-regulate Ca_V1.2 containing the full-length α_1C, Ca_V1.2-α_1C (24–26). However, robust β-AR–induced up-regulation of Ca²⁺ currents was observed in oocytes injected with total heart RNA (27, 28), suggesting the necessity of an auxiliary protein, the “missing link” (24, 25). Interestingly, partial regulation was observed with dCT-truncated α_1C (16, 29). Intracellular injection of cAMP or PKA-CS in Xenopus oocytes caused a modest (30 to 40%) up-regulation of Ca_V1.2, containing a dCT-truncated α_1C, Ca_V1.2-α_1CΔ1821 (29). This regulation required the presence of the initial segment of the long-NT of α_1C but did not involve Ca_Vβ subunit. We proposed that this mechanism might account for part of the adrenergic regulation of Ca_V1.2 in the heart (29). Normally adrenergic stimulation in cardiomyocytes increases the Ca²⁺ current two- to threefold; thus, a major part of the regulation has remained unexplained.Recently, Liu et al. identified Rad as the “missing link” in PKA regulation of Ca_V1.2 (20). Rad is a member of the Ras-related GTP-binding protein subfamily (RGK) that inhibit high voltage-gated calcium channels Ca_V1 and Ca_V2 (30). Rad tonically inhibits Ca_V1.2, largely via an interaction with Ca_Vβ (31, 32). Ablation of Rad in murine heart was shown to increase basal Ca_V1.2 activity and rendered the channel insensitive to β-AR regulation, probably through a “ceiling” effect (33, 34). Liu et al. (20) reconstituted a major part of the Ca_V1.2 regulation cascade, initiated by forskolin-activated adenylyl cyclase in mammalian cells, ultimately attaining an approximately twofold increase in Ca²⁺ current. The regulation required phosphorylation of Rad, the presence of Ca_Vβ, and the interaction of Ca_Vβ with the cytosolic loop I of α_1C, suggesting that PKA phosphorylation of Rad reduces its interaction with Ca_Vβ and relieves the tonic inhibition of Ca_V1.2 (20, 35).Importantly, the complete adrenergic cascade, starting with β-AR activation, has not yet been heterologously reconstituted for Ca_V1.2. Also, the relation between the Rad-dependent regulation and the regulation reported in our previous study (29) is not clear. Here, we utilized the Xenopus oocyte heterologous expression system and successfully reconstituted the entire β-AR cascade. We demonstrate two distinct pathways of PKA modulation of Ca_V1.2 (Rad dependent and Rad independent) and characterize the roles of NT and CT of α_1C, β_2b, and Rad in the adrenergic modulation of cardiac Ca_V1.2 channels. Reproducing the complete β-AR cascade in a heterologous expression system will promote the identification and characterization of intracellular proteins that regulate the cascade, eventually assisting efforts to develop therapies to treat heart failure and other catecholamine-induced cardiac pathologies.     

Superelastic oxide micropillars enabled by surface tension–modulated 90° domain switching with excellent fatigue resistance

Superelastic materials capable of recovering large nonlinear strains are ideal for a variety of applications in morphing structures, reconfigurable systems, and robots. However, making oxide materials superelastic has been a long-standing challenge due to their intrinsic brittleness. Here, we fabricate ferroelectric BaTiO₃ (BTO) micropillars that not only are superelastic but also possess excellent fatigue resistance, lasting over 1 million cycles without accumulating residual strains or noticeable variation in stress–strain curves. Phase field simulations reveal that the large recoverable strains of BTO micropillars arise from surface tension–modulated 90° domain switching and thus are size dependent, while the small energy barrier and ultralow energy dissipation are responsible for their unprecedented cyclic stability among superelastic materials. This work demonstrates a general strategy to realize superelastic and fatigue-resistant domain switching in ferroelectric oxides for many potential applications.

Superelastic materials are capable of recovering large amount of nonlinear “plastic” strains, way beyond their linear elastic regimes (1–4). They are ideal for a variety of applications from morphing structures, reconfigurable systems, to robots (5–8). The effects have traditionally been associated with macroscopically compliant/ductile rubbers (2) or microscopically phase-transforming shape memory alloys (SMAs) (7–11). The only macroscopically brittle oxide recently discovered to be superelastic is ZrO₂-based micropillars or particles (12–20), which is realized via austenite-martensite phase transformation similar to SMAs. Although ultimate strengths approaching the theoretical limit have been demonstrated in nanoscale samples (21, 22), long fatigue life is elusive, which is arguably more important for most applications. As a matter of fact, poor fatigue life has been a long-standing challenge for oxide ceramics in general (23, 24). Even for ductile SMAs that enjoy excellent fatigue life, irrecoverable residual strains gradually accumulate over cycling, leading to substantial variations in stress–strain curves at different cycles (9, 10, 25). We overcome these difficulties by reporting superelastic barium titanate (BaTiO₃ [BTO]) micropillars enabled by surface tension–modulated 90° domain switching, which exhibit excellent fatigue resistance, while bulk BTO crystals or ceramics are rather brittle. The demonstration of over one million cycles of loading and unloading without accumulating residual strains or noticeable variation in stress–strain curves is unprecedented among superelastic materials.BTO is a ferroelectric oxide exhibiting modest piezoelectric strains around 0.1 to 0.2% (26) and fracture toughness of ∼1 MPa ⋅ m^1/2, and thus it is quite brittle (27). Considerable research efforts have been devoted to enhancing its electric field–induced strain via 90° ferroelectric domain switching (28–30). However, the process is often irreversible, and external mechanisms such as restoring force (28, 29) and internal mechanisms such as defect pinning (30) have to be invoked to make the electrostrain recoverable. Nevertheless, it hints at the possibility of BTO being made superelastic by taking advantage of the stress-induced 90° domain switching (6). Earlier works suggest that surface tension induces an in-plane compressive stress that favors the axial polarization in one-dimensional ferroelectrics at small size (31, 32), which may provide the necessary restoring mechanism for the stress-switched domains. Thus, if a compressive axial force is applied, reversible domain switching may occur during unloading, leading to superelasticity. To verify this hypothesis, we fabricated single-crystalline BTO micropillars from [001]-oriented bulk crystals (SI Appendix, Fig. S1A) via focused ion beam (FIB), as detailed in Materials and Methods and SI Appendix, Fig. S1B. The diameters (Φ) of the micropillars range from 0.5 μm to 5 μm, with their height to diameter ratio fixed at 3. No visible defects can be seen from the scanning electron microscopy (SEM) images of these micropillars shown in Fig. 1 A–D, and their surfaces appear to be quite smooth, suggesting that no apparent damages are induced by FIB.Open in a separate windowFig. 1.Superelastic BTO micropillars below a critical size. (A–D) SEM images of the micropillars with Φ = 5, 3, 2, and 0.5 μm. (E–G) The first and second cycles of stress–strain curves for BTO micropillars with Φ = 5, 2, and 0.5 μm. (H) S_r/S_max and ΔW/W_max during the first cycle for BTO micropillars of different diameters. Here, S_r and S_max denote the residual strain and the maximum strain (SI Appendix, Fig. S6A), while ΔW and W_max are energy dissipated and stored in the first cycle, respectively (SI Appendix, Fig. S6F).     

The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1–infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1–infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)₃ neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

Membrane fusion between HIV-1 and host cells is mediated by the viral envelope glycoprotein (Env), a trimer consisting of the gp120 and gp41 subunits. Upon interaction with cellular receptors, Env undergoes a dramatic conformational change and forms the prehairpin intermediate (PHI) (1–3), in which the fusion peptide region at the amino terminus of gp41 inserts into the cell membrane. In the PHI, the N-heptad repeat (NHR) region of gp41 is exposed and forms a stable, three-stranded α-helical coiled coil. Subsequently, the PHI resolves when the NHR and the C-heptad repeat (CHR) regions of gp41 associate to form a trimer-of-hairpins structure that brings the viral and cell membranes into proximity, facilitating membrane fusion (Fig. 1).Open in a separate windowFig. 1.HIV-1 membrane fusion. The surface protein of the HIV-1 envelope is composed of the gp120 and gp41 subunits. After Env binds to cell-surface receptors, gp41 inserts into the host cell membrane and undergoes a conformational change to form the prehairpin intermediate. The N-heptad repeat (orange) region of gp41 is exposed in the PHI and forms a three-stranded coiled coil. To complete viral fusion, the PHI resolves to a trimer-of-hairpins structure in which the C-heptad repeat (blue) adopts a helical conformation and binds the NHR region. Fusion inhibitors such as enfuvirtide bind the NHR, preventing viral fusion by inhibiting formation of the trimer of hairpins (1–3). The membrane-proximal external region (red) is located adjacent to the transmembrane (TM) region of gp41.The NHR region of the PHI is a validated therapeutic target in humans: the Food and Drug Administration (FDA)-approved drug enfuvirtide binds the NHR and inhibits viral entry into cells (4, 5). Various versions of the three-stranded coiled coil formed by the NHR have been created and used as vaccine candidates in animals (6–10). The neutralization potencies of these antisera, as well as those of anti-NHR monoclonal antibodies (mAbs) (11–15), are modest and mostly limited to HIV-1 isolates that are highly sensitive to antibody-mediated neutralization [commonly referred to as tier-1 viruses (16)]. These results have led to skepticism about the PHI as a vaccine target.Earlier studies showed that the neutralization activities of mAbs that bound another region of gp41, the membrane-proximal external region (MPER) (Fig. 1), were enhanced as much as 5,000-fold in cells expressing FcγRI (CD64) (17, 18), an integral membrane protein that binds the Fc portion of immunoglobulin G (IgG) molecules with high (nanomolar) affinity (19, 20). This effect was not attributed to phagocytosis and occurred when the cells were preincubated with antibody and washed before adding virus (17, 18). Since the MPER is a partially cryptic epitope that is not fully exposed until after Env engages with cellular receptors (21, 22), these results suggest that by binding the Fc region, FcγRI provides a local concentration advantage for MPER mAbs at the cell surface that enhances viral neutralization (17, 18). While not expressed on T cells, FcγRI is expressed on macrophages and dendritic cells (23), which are present at mucosal surfaces and are implicated in sexual HIV-1 transmission and the early establishment of HIV-1 infection (22–34).Here we investigated whether FcγRI expression also potentiates the neutralizing activity of antibodies targeting the NHR, since that region, like the MPER, is preferentially exposed during viral fusion. We found that D5, a well-characterized anti-NHR mAb (11, 12), inhibits HIV-1 infection ∼5,000-fold more potently in TZM-bl cells expressing FcγRI (TZM-bl/FcγRI cells) than in TZM-bl cells that do not. Further, while antisera from guinea pigs immunized with (ccIZN36)₃, an NHR-based vaccine candidate (7), displayed weak neutralizing activity in TZM-bl cells, they exhibited enhanced neutralization in TZM-bl/FcγRI cells, including against some tier-2 HIV-1 isolates that are more resistant to antibody-mediated neutralization (16) and that serve as benchmarks for antibody-based vaccine efforts. These results indicate that FcγRI can play an important role in neutralization by antibodies that target the PHI. Since these receptors are expressed on cells prevalent at mucosal surfaces thought to be important for sexual HIV-1 transmission, our results motivate vaccine strategies that harness this potentiating effect.     

Geometrical manipulation of complex supramolecular tessellations by hierarchical assembly of amphiphilic platinum(II) complexes

Here we report complex supramolecular tessellations achieved by the directed self-assembly of amphiphilic platinum(II) complexes. Despite the twofold symmetry, these geometrically simple molecules exhibit complicated structural hierarchy in a columnar manner. A possible key to such an order increase is the topological transition into circular trimers, which are noncovalently interlocked by metal···metal and π–π interactions, thereby allowing for cofacial stacking in a prismatic assembly. Another key to success is to use the immiscibility of the tailored hydrophobic and hydrophilic sidechains. Their phase separation leads to the formation of columnar crystalline nanostructures homogeneously oriented on the substrate, featuring an unusual geometry analogous to a rhombitrihexagonal Archimedean tiling. Furthermore, symmetry lowering of regular motifs by design results in an orthorhombic lattice obtained by the coassembly of two different platinum(II) amphiphiles. These findings illustrate the potentials of supramolecular engineering in creating complex self-assembled architectures of soft materials.

Tessellation in two dimensions (2D) is a very old topic in geometry on how one or more shapes can be periodically arranged to fill a Euclidean plane without any gaps. Tessellation principles have been extensively applied in decorative art since the early times. In natural sciences, there has been a growing attention on creating ordered structures with increasingly complex architectures inspired by semi-regular Archimedean tilings (ATs) and quasicrystalline textures on account of their intriguing physical properties (1–5) and biological functions (6). Recent advances in this regard have been achieved in various fields of supramolecular science, including the programmable self-assembly of DNA molecules (7), coordination-driven assembly (8–10), supramolecular interfacial engineering (11–13), crystallization of organic polygons (14, 15), colloidal particle superlattices (16), and other soft-matter systems (17–20). Moreover, tessellation in 2D can overcome the topological frustration to generate complex semi- or non-regular patterns by using geometrically simple motifs. As exemplified by the self-templating assembly of spherical soft microparticles (21), a vast array of 2D micropatterns encoding non-regular tilings, such as rectangular, rhomboidal, hexagonal, and herringbone superlattices were obtained by layer-by-layer strategy at a liquid–liquid interface. Tessellation principles have also been extended to the self-assembly of giant molecules in three dimensions (3D). Superlattices with high space-group symmetry (Im3¯m, Pm3¯n, and P4₂/mnm) were reported in dendrimers and dendritic polymers by Percec and coworkers (22–24). Recently, Cheng and coworkers identified the highly ordered Frank–Kasper phases obtained from giant amphiphiles containing molecular nanoparticles (25–28). Despite such advancements made in the field of soft matter, an understanding of how structural ordering in supramolecular materials is influenced by the geometric factors of its constituent molecules has so far remained elusive.In light of these developments and the desire to explore the supramolecular systems, square-planar platinum(II) (Pt^II) polypyridine complexes may serve as an ideal candidate for model studies not only because of their intriguing spectroscopic and luminescence properties (29, 30), but also because of their propensity to form supramolecular polymers or oligomers via noncovalent Pt···Pt and π–π interactions (31–39). Although rod-shaped and lamellar structures are the most commonly observed in the self-assembly of planar Pt^II complexes (34–39), 2D-ordered nanostructures, such as the hexagonally packed columns (31, 40) and honeycomb-like networks (41–43), were recently first demonstrated by us.Herein, we report a serendipitous discovery of a C_2h-symmetric Pt^II amphiphile (Fig. 1A) that can hierarchically self-assemble into a 3D-ordered nanostructure with hexagonal geometry. Interestingly, this structurally anisotropic molecule possibly undergoes topological transition and interlocks to form its circular trimer by noncovalent Pt···Pt and π–π interactions (Fig. 1B). The resultant triangular motif is architecturally stabilized and preorganized for one-dimensional (1D) prismatic assembly (Fig. 1C). Together with the phase separation of the tailored hydrophobic and hydrophilic sidechains, an unusual and unique 3D hexagonal lattice is formed (Fig. 1D), in which the Pt centers adopt a rare rhombitrihexagonal AT-like order. Finally, the nanoarchitecture develops in a hierarchical manner on the substrate due to the homogeneous nucleation (Fig. 1E).Open in a separate windowFig. 1.Hierarchical self-assembly of Pt^II amphiphile into hexagonal ordering. (A) Space-filling (CPK) model of a C_2h-symmetric Pt^II amphiphile (1). All of the hydrogen atoms and counterions are omitted for clarity. (B) CPK representations of possible models of regular triangular, tetragonal, pentagonal, and hexagonal motifs formed with Pt···Pt and π–π stacking. These motifs possess a hydrophilic core (red) with various diameters wrapped by a hydrophobic shell comprising long alkyl chains (gray). (C) CPK representation of a 1D prismatic structure consisting of circular trimers with long-range Pt···Pt and π–π stacking. (D) CPK representation of a 3D columnar lattice constructed by the prismatic assemblies adopting a rare rhombitrihexagonal AT-like order. With the assistance of the phase separation, the hydrophobic domain serves as a discrete column associated with six prismatic neighbors. (E) Schematic representation of the nanoarchitecture with homogeneous orientation.     

Citramalate synthase yields a biosynthetic pathway for isoleucine and straight- and branched-chain ester formation in ripening apple fruit

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple (Malus × domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. ¹³C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in “Jonagold” fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine³⁸⁷ (MdCMS_1) or glutamate³⁸⁷ (MdCMS_2). Glutamate³⁸⁷ was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate³⁸⁷ produced a very low proportion of 2-methylbutanol- and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.

Esters are aroma impact compounds produced by many fruits and contribute notably to the sensory quality of apple (Malus × domestica) fruit, accounting for 80 to 95% of the total volatiles emitted (1). The esters hexyl acetate, butyl acetate, and 2-methylbutyl acetate are abundantly produced and considered to confer typical apple aroma characteristics (2, 3), which are perceived as “fruity” and “floral.” Volatile esters produced in apple fruit are largely composed of either straight-chain (SC) or branched-chain (BC) alkyl (alcohol-derived) and alkanoate (acid-derived) groups, which typically possess one to eight carbons (1). The final step of ester formation is the condensation of an alcohol and a CoA thioester by alcohol acyltransferase (AAT) (4). Surprisingly, despite the importance of aroma in fruit consumption, the biochemistry of ester formation is poorly understood.It has been suggested that ester precursors are produced primarily by degradative processes and that BC ester precursors, in particular, are derived from branched-chain amino acid (BCAA) degradation (5–9). In apples, isoleucine accumulates during apple fruit ripening, but valine and leucine do not (10–12). Correspondingly, esters related to isoleucine metabolism predominate, while those from valine can be detected only occasionally and usually at low levels, and no esters are produced from the leucine pathway (9, 13, 14). In plants, isoleucine is normally synthesized from threonine based on evidence for autotrophy in Nicotiana plumbaginifolia (15, 16). Threonine is deaminated to α-ketobutyrate by threonine deaminase (TD) (17), and α-ketobutyrate is subsequently metabolized to α-keto-β-methylvalerate, the isoleucine precursor, by three enzymes (Fig. 1). These same three enzymes form α-ketoisovalerate from pyruvate in the synthesis pathway for valine. Leucine synthesis begins with the valine precursor α-ketoisovalerate to form the leucine precursor α-ketoisocaproate.Open in a separate windowFig. 1.Branched-chain amino acid metabolism, proposed citramalate pathway, and routes to ester biosynthesis. Citramalate-dependent pathway (in red) and its contribution to straight- and branched-chain ester biosynthesis. Adapted from Sugimoto et al. (40). The long dashed lines indicate feedback inhibition. The short dashed lines indicate not all reactions shown. Abbreviations: AAT, alcohol acyl-CoA transferase; ADH, alcohol dehydrogenase; BCAT, branched-chain amino transferase; BCKDC, branched-chain α-ketoacid decarboxylase; BCKDH, branched-chain α-ketoacid dehydrogenase; CMS, citramalate synthase; IPMDH, 3-isopropylmalate dehydrogenase; IPMI, 2-isopropylmalate isomerase; IPMS, 2-isopropylmalate synthase. An asterisk indicates the gene has been previously found in bacteria, but not in plants. The double asterisk indicates activity previously given the trivial name of 2-ethylmalate synthase (77). Hydrogens in carbon–hydrogen bonds are not shown. Carbons derived from the C-1 and C-2 positions of acetyl-CoA are, respectively, indicated with open and solid symbols adjacent to the carbon atoms.The final reaction in the synthesis of isoleucine, valine, and leucine involves branched-chain aminotransferase (BCAT), which catalyzes a freely reversible reaction (Fig. 1). BC esters can be produced from exogenously supplied BCAAs, but also by the application of BC α-ketoacids (α-KEAs) (6). Given that BC α-KEAs are in approximate equilibrium with their respective BCAAs (18), it may be reasonable to expect that, for apple, the pool of isoleucine roughly mirrors the pool of its respective BC α-KEA. Therefore, the accumulation of isoleucine in apples during ripening may well be an indication of the content of its precursor, α-keto-β-methylvalerate. Furthermore, α-keto-β-methylvalerate is ultimately the direct precursor to the BC ester 2-methylbutyl acetate, an important aroma impact compound for apple (1).Biosynthesis of all three BCAAs is responsive to feedback regulation. TD is inhibited by isoleucine, although this inhibition is antagonized by valine; acetohydroxyacid synthase is principally inhibited by valine and leucine; and 2-isopropylmalate synthase (IPMS) is inhibited by leucine (19–21). Given that isoleucine biosynthesis is under feedback regulation, the explanation for the exclusive accumulation of this amino acid in ripening apple fruit is not obvious. Sugimoto et al. (12) used this evidence to propose the existence of an alternative pathway for α-ketobutyrate formation in ripening apple fruit, whose first step involves the formation of citramalate.The citramalate pathway has been described in several strains of bacteria for isoleucine biosynthesis (22–25). In this pathway, acetyl-CoA and pyruvate are substrates for the formation of citramalate by citramalate synthase (CMS) (Fig. 1). Several bacteria form (R)-citramalate, whereas yeast and apple form (S)-citramalate (26, 27). CMS is closely related to IPMS, which belongs to an acyltransferase family (EC 2.3.3). The acyltransferase family also includes citrate synthase, homocitrate synthase, malate synthase, and methylthioalkylmalate synthase (MAM). Each differs in substrate specificity, preferring, respectively, oxaloacetate, α-ketoglutarate, glyoxylate, α-ketoisovalerate, and various ω-methylthio-α-ketoalkanoates (28, 29).In Leptospira interrogans, LiCMS (UniProtKB-{"type":"entrez-protein","attrs":{"text":"Q8F3Q1","term_id":"81748238","term_text":"Q8F3Q1"}}Q8F3Q1) protein has a sequence similar to Mycobacterium tuberculosis MtIPMS, but unlike IPMS, its activity is specific to pyruvate as the α-KEA substrate (30, 31). In Arabidopsis, four genes in the IPMS family (IPMS1 [At1g18500], IPMS2 [At1g74040], MAM1 [At5g23010], and MAM3 [At5g23020]) have been characterized (28, 32, 33). The amino acid sequence identity is ∼60% between AtIPMS and AtMAM proteins (32) and the most significant difference is the presence of an additional 130-aa sequence in the C-terminal region in AtIPMS. This domain, called the “R-region,” is involved in leucine feedback inhibition in the yeast IPMS protein (LEU4) (34). AtIPMS and LiCMS enzymes are inhibited by leucine (32) and isoleucine (35), respectively; however, the lack of the R-region in AtMAM eliminates leucine feedback inhibition (36).CMS proteins in Methanococcus jannaschii (UniProtKB-{"type":"entrez-protein","attrs":{"text":"Q58787","term_id":"2492795","term_text":"Q58787"}}Q58787) and L. interrogans have been characterized for their activity and specificity (30, 37). In yeast, CMS activity is evident in both Saccharomyces cerevisiae (38) and Saccharomyces carlsbergensis (26), but no nucleotide or amino acid sequence for CMS has been identified as yet in the yeast genome database. In plants, Kroumova and Wagner (39) reported the involvement of one-carbon fatty acid biosynthesis (1-C FAB) in the formation of sugar esters in some (e.g., tobacco [Nicotiana tabacum] and petunia [Petunia × hybrida]), but not all, members of the Solanaceae and suggested that an α-KEA elongation pathway initiated by the condensation of acetyl-CoA and pyruvate enables 1-C FAB. However, there has been no molecular characterization of the entry point into the pathway via CMS, for example, in these or other plant species, nor, in fact, in any eukaryote.Although previous works have suggested that catabolic pathways are primarily responsible for ester biosynthesis (5–8), the lack of supportive molecular and biochemical data suggests that a reassessment of this conceptual model is appropriate. The objective of this work was to evaluate whether the citramalate pathway operates in specialized plant organs like apple fruit and whether it contributes to the synthesis of BC and SC esters. Herein, we identify and characterize two MdCMS alleles and their translated protein isomers, demonstrate the presence of an active citramalate pathway in apple that includes α-KEA elongation, and link these elements to ester biosynthesis. Our work builds upon that of Hulme (27), who originally identified citramalate from plant (apple) extracts, and confirms the hypothesis of Sugimoto et al. (12, 40) regarding the existence of a citramalate pathway in plants that, in apple, contributes to ester biosynthesis.     

Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

The radical S-adenosylmethionine (rSAM) enzyme superfamily is the largest known in nature, with over 570,000 annotated and predominantly uncharacterized members spanning all domains of life (1–4). The uniting feature of rSAM enzymes is a [4Fe-4S] cluster, usually bound by a CX₃CX₂C motif that catalyzes reductive cleavage of SAM to form L-Met and a strongly oxidizing 5′-deoxyadenosyl radical (5′-dA•) (5–7). Recent studies on a suite of rSAM enzymes have revealed the presence of a previously unknown organometallic intermediate in this process, termed Ω, in which the 5′-C of 5′-dA• is bound to the unique iron of the [4Fe-4S] cluster (Fig. 1A) (8, 9). Homolysis of the Fe–C bond ultimately liberates 5′-dA•, which abstracts a hydrogen atom from substrate to initiate a profoundly diverse set of chemical reactions in both primary and secondary metabolism, including DNA, cofactor, vitamin, and antibiotic biosynthesis (5, 10–13).Open in a separate windowFig. 1.(A) Accepted scheme for radical initiation in rSAM enzymes. (B) X-ray crystal structure of SuiB (PDB ID: 5V1T). The RS domain, SPASM domain, and RiPP recognition element are rendered blue, green, and pink, respectively. [4Fe-4S] clusters are shown as spheres with the distances separating them indicated. (C) Lys–Trp cross-link formation (20) catalyzed by SuiB. The carbon–carbon bond installed by SuiB is shown in red. (D and E) Previously proposed EAS (D) and rEAS (E) mechanisms for SuiB-catalyzed Lys–Trp cross-link formation.Of the 570,000 rSAM enzyme superfamily members, over a quarter (∼165,000 genes from the Enzyme Function Initiative-Enzyme Similarity Tool) possess C-terminal extensions, called SPASM and twitch domains, which bind auxiliary Fe-S clusters (4, 14–19). The SPASM domain typically binds two auxiliary Fe-S clusters and is named after the rSAM enzymes involved in the synthesis of subtilosin, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin. The twitch domain is a truncated SPASM domain and only binds one auxiliary cluster (15). Despite the wide prevalence of these domains and the characterization of several different SPASM/twitch rSAM enzymes by spectroscopic and structural studies, direct evidence for their catalytic function(s) has remained elusive.We previously performed functional and structural characterization on the SPASM rSAM enzyme SuiB (Fig. 1B), which is involved in the biosynthesis of a ribosomal peptide natural product in human and mammalian microbiome streptococci (14, 20–22). SuiB introduces an unusual carbon–carbon bond onto its substrate peptide, SuiA, between the sidechains of Lys2 and Trp6 (Fig. 1C). The mechanism for this transformation is of broader relevance, as a number of enzymes, such as RrrB, PqqE, and MqnC (2, 23, 24), are known to join unactivated aliphatic and aromatic carbons to generate sp³-sp² cross-links. A general mechanistic paradigm for this class of transformations is not yet available. For SuiB, two pathways have been proposed (20), one through a typical electrophilic aromatic substitution (EAS) mechanism, which is involved in other enzyme-catalyzed indole modifications, such as indole prenylation or flavin adenine dinucleotide (FAD)-enzyme-dependent indole chlorination (25–27). In this pathway, the 5′-dA• generates an alkyl radical, which upon a second one-electron oxidation, creates an α,β-unsaturated amide electrophile with which the indole sidechain reacts via Michael addition (Fig. 1D). Lanthionine cross-links observed in diverse lanthipeptides are built by this general scheme, though via heterolytic chemistry, with Cys acting as the nucleophile (28, 29). Alternatively, a radical electrophilic aromatic substitution (rEAS) reaction has been proposed, wherein the alkyl radical, formed by 5′-dA•, would react with the indole sidechain to generate a radical σ complex, a cross-linked Lys–Trp radical (Lys–Trp•), which upon oxidation and rearomatization would yield product (Fig. 1E). In both mechanisms, AuxI is proposed as an oxidant. Although this role for an rSAM auxiliary cluster has been previously suggested (30, 31), it has yet to be directly demonstrated experimentally. Mechanistic studies have favored the rEAS pathway (20); however, intermediates in the reaction of SuiB and enzymes that catalyze similar reactions have not yet been detected (15).In the current work, we sought to differentiate between the proposed mechanisms by trapping intermediates in the catalytic cycle of SuiB and characterizing them using electron paramagnetic resonance (EPR) spectroscopy. We report observation of three transient reaction intermediates, most importantly the sought-after Lys–Trp•, which is fundamentally different from previously characterized Trp radicals, as it is cross-linked and carries an indole tetrahedral center. We also provide evidence for AuxI as the oxidant of the Lys–Trp• intermediate as well as insights into redox potential changes of Fe-S clusters in SuiB that accompany SuiA binding. Together, our findings support the rEAS pathway for formation of the sp³-sp² cross-link and carry important implications for other enzymes that catalyze related transformations.     

Multiple deprotonation paths of the nucleophile 3′-OH in the DNA synthesis reaction

DNA synthesis by polymerases is essential for life. Deprotonation of the nucleophile 3′-OH is thought to be the obligatory first step in the DNA synthesis reaction. We have examined each entity surrounding the nucleophile 3′-OH in the reaction catalyzed by human DNA polymerase (Pol) η and delineated the deprotonation process by combining mutagenesis with steady-state kinetics, high-resolution structures of in crystallo reactions, and molecular dynamics simulations. The conserved S113 residue, which forms a hydrogen bond with the primer 3′-OH in the ground state, stabilizes the primer end in the active site. Mutation of S113 to alanine destabilizes primer binding and reduces the catalytic efficiency. Displacement of a water molecule that is hydrogen bonded to the 3′-OH using the 2′-OH of a ribonucleotide or 2′-F has little effect on catalysis. Moreover, combining the S113A mutation with 2′-F replacement, which removes two potential hydrogen acceptors of the 3′-OH, does not reduce the catalytic efficiency. We conclude that the proton can leave the O3′ via alternative paths, supporting the hypothesis that binding of the third Mg²⁺ initiates the reaction by breaking the α–β phosphodiester bond of an incoming deoxyribonucleoside triphosphate (dNTP).

DNA polymerases catalyze the incorporation of deoxyribonucleoside monophosphate (dNMP) into an existing primer after binding a deoxyribonucleoside triphosphate (dNTP) complementary to a templating base. The catalytic process is thought to begin with deprotonation of the primer 3′-OH by a general base, continue by nucleophilic attack of the α-phosphate of the dNTP leading to a new phosphodiester bond, and finish with release of pyrophosphate after its protonation by a general acid (Fig. 1A) (1). This mechanism has long been established to require two Mg²⁺ ions (A and B) (2). However, visualizing the reaction intermediates using time-resolved X-ray crystallography reveals little movement of the protein but a third and transiently bound Mg²⁺ ion necessary for the DNA synthesis reaction (3, 4). Whether the reaction is driven by the third Mg²⁺ ion or the nucleophile 3′-OH, the identity of a general base responsible for deprotonating the primer 3′-OH remains unclear.Open in a separate windowFig. 1.DNA synthesis reaction. (A) Deprotonation and activation of the primer-end 3′-OH for the nucleophilic attack is hypothesized to be the first step. (B) In the three–Mg²⁺-ion catalysis, the first step appears to break the phosphodiester bond between the α- and β-phosphates of the dNTP. (C) Structural superposition of the reaction-ready (RS) and product state of the WT enzyme in extending the dT primer. The two Mg²⁺ ions (A and B sites) are bound and the 3′-OH is hydrogen-bonded to the transient WatN, which in turn is linked to the bulk solvent via another water molecule. The third Mg²⁺ (C site) is observed only with the products. (D) In the ground state with one Ca²⁺ (green sphere), the 3′-OH is hydrogen-bonded with S113 and not aligned for the inline nucleophilic attack in the absence of the A-site Mg²⁺. The aligned 3′-OH in the RS is shown as semitransparent sticks as a reference. (E) The transient WatN (circled in orange dashes) appears only in the RS and would clash with the C2′ in the GS (wheat) and PS (blue) as indicated by double arrowheads. (F) Sequence alignment of active-site residues around S113 (highlighted in red) of human Pol η, Saccharomyces cerevisiae Pol η (yeast), human Pol ι, human Pol κ, S. cerevisiae Rev1, Escherichia coli Pol IV, and Sulfolobus solfataricus Dpo4. The conserved secondary structures are shown above the sequences.Crystal structures of various DNA polymerases bound to DNA and dNTP, known as ternary complexes, reveal a conserved catalytic center with two Mg²⁺ ions coordinated by the conserved catalytic carboxylates, three phosphates of an incoming dNTP, and the primer 3′-OH (Fig. 1C) (5, 6). DNA polymerase has been likened to a right hand with the palm domain containing the catalytic residues, the finger domain closing on top of the nascent or replicating base pair, and the thumb domain binding the upstream DNA (product) duplex (7, 8). Many DNA polymerases undergo a large conformational change involving closing of the finger domain upon binding a correct dNTP (8), which was proposed to be a rate-limiting step (9). However, the rate of finger domain closing is much faster than the rate of chemical reaction (10–13). Moreover, the finger domain of the Y-family DNA polymerases is already closed even in the absence of an incoming nucleotide (14). Although intradomain motions have been implicated in one example (15), the rate-limiting step is probably the chemical reaction (3, 4, 16, 17).If nucleophilic attack is the first step, deprotonation of the primer 3′-OH by a general base would be essential to initiate the reaction (Fig. 1A). In the more than three decades since the first DNA polymerase structure was determined (7), no residues other than the conserved carboxylates that coordinate the two Mg²⁺ ions have been found to eliminate the catalytic activity when mutated. Computationally, the conserved carboxylates (1, 18) and the incoming nucleotide together with the water molecule bound to the A-site Mg²⁺ (WatA) have each been suggested to deprotonate the 3′-OH (12, 19–21). As the carboxylates and dNTPs are necessary for Mg²⁺ binding and the synthesis reaction, their role in deprotonation is nearly impossible to be experimentally tested.A third Mg²⁺ ion (occupying the C site) has been observed to transiently bind to dNTP in the reactions catalyzed by different DNA polymerases (3, 22–24). Unlike the A- and B-site Mg²⁺ ions, which are coordinated by the catalytic carboxylates, the C-site Mg²⁺ does not contact the enzyme at all. Its low affinity (k_d) matches the minimal Mg²⁺ concentration required for catalysis (3, 4). When two canonical Mg²⁺ ions are bound and reactants are aligned for the inline nucleophilic attack, the third Mg²⁺ ion does not bind readily (3, 4). Only when products are formed is the third Mg²⁺ ion observed to bind between the product DNA and pyrophosphate with four additional water ligands. Yet without the third Mg²⁺, no product can form (4). Its binding is thermal energy (temperature)-dependent, and concurrent with the product formation. The third Mg²⁺ ion may drive dNMP incorporation by breaking the α–β phosphodiester bond of dNTP and pushing the α-phosphate toward the 3′-OH for the new bond formation (4) (Fig. 1B). With the C-site Mg²⁺, deprotonating the 3′-OH is likely favored and does not need a strong general base.The in crystallo analysis of the DNA synthesis reaction catalyzed by human polymerase (Pol) η reveals three reaction states and two potential candidates to deprotonate the nucleophile. Initial dNTP binding with one divalent cation only (B site) leads to the ground state (GS) (Fig. 1D). Binding of the second Mg²⁺ converts the GS to the reactive state (RS), in which the 3′-OH is aligned for the inline nucleophilic attack, and appearance of the third Mg²⁺ (C site) is coupled with the product formation (product state; PS) (3, 4) (Fig. 1 C and E). In the GS, the 3′-OH is within 2.7 Å and hydrogen-bonded to the hydroxyl group of S113 (3). Although S113 is highly conserved among the Y-family polymerases (Fig. 1F) and may facilitate deprotonation of the 3′-OH, removal of the hydroxyl group by mutating S113 to Ala in human Pol η reduces the catalytic efficiency but does not eliminate catalysis (3). In the RS, the 3′-OH is 2.7 Å from a water molecule, which is termed WatN (N for nucleophile) and hypothesized to shuttle the proton off the 3′-OH to the bulk solvent (3). However, preliminary kinetic analysis showed that displacement of the water molecule by the 2′-OH of a ribonucleotide at the primer end leaves catalysis of DNA synthesis unaltered (3).Here we investigate in detail how the 3′-OH is deprotonated in the reaction catalyzed by human Pol η. Combining S113A mutant Pol η (S113A) and modified nucleotides at the primer 3′ end, we measured how the altered environment of the 3′-OH perturbed the steady-state kinetics and reaction process in crystallo. In addition, static structures and dynamics simulation are employed to explain observed kinetic parameters. In contrast to the hypothesis of a specific general base, we find that the proton of the 3′-OH can depart via multiple paths.     

NMR unveils an N-terminal interaction interface on acetylated-α-synuclein monomers for recruitment to fibrils

Amyloid fibril formation of α-synuclein (αS) is associated with multiple neurodegenerative diseases, including Parkinson’s disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of αS fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-αS (Ac-αS) monomers with seeding-competent Ac-αS fibrils and seeding-incompetent off-pathway oligomers to identify Ac-αS monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway αS oligomers can act as an auto-inhibitor against αS fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation.

Amyloid formation of the intrinsically disordered protein (IDP) α-Synuclein (αS) is closely associated with the pathogenesis of a variety of neurodegenerative disorders, including Parkinson’s disease (PD), dementia with Lewy bodies, and multiple-systems atrophy (1). The pathology of αS is still not clearly understood, as multiple species along the fibril aggregation pathway, including on-pathway oligomeric intermediates and end-stage fibrils, have been demonstrated to be toxic to neurons (2–8). Abrogation of these toxic species is extremely challenging, in part due to the amyloid seeding process, which is a complex mechanism by which endogenous monomers interact with existing fibril seeds to facilitate additional fibril formation. The mechanism involves multiple microscopic steps, including elongation of fibril ends and surface-mediated secondary nucleation (9–11). Furthermore, disease progression has become increasingly linked to prion-like cell-to-cell propagation of αS aggregates, in which αS oligomers and fibrils are transmitted to neighboring neurons (12, 13). Here, the invading aggregates seed amyloid formation of endogenous monomers to accelerate production of toxic species (3). Thus, disruption of the process by which αS aggregates seed proliferative amyloid formation has become an intriguing target for inhibition of pathological αS self-assembly and may reveal new therapeutic approaches against PD.Under amyloid fibril formation conditions in vitro, αS monomers self-assemble along multiple pathways to mature into various species of stable off-pathway oligomers, transient on-pathway oligomers that convert into amyloid fibrils, fragmented fibril seeds and mature fibrils, creating a complex, heterogeneous environment (Fig. 1A). While the inherent structural inhomogeneity and transient nature of on-pathway oligomers makes them extremely challenging to isolate and characterize, off-pathway oligomers are stable and can be isolated by size exclusion chromatography (SEC). Multiple species of stable, off-pathway αS oligomers under various conditions have been previously isolated (5, 14–16). Lorenzen et al. isolated two forms of stable αS oligomers from amyloid-promoting conditions and determined the structure of the smaller oligomers to consist of ∼30 monomers oriented in an ellipsoid shape, with an outer cloud of flexible and disordered molecules, based on small angle X-ray scattering (SAXS) (14) (Fig. 1A). How these stable oligomers impact amyloid seeding and growth is not well understood. Determining how stable oligomers influence amyloid aggregation mechanisms may provide insight into pathological seeding processes and aid in discovery of novel therapeutic approaches against amyloid seeding.Open in a separate windowFig. 1.In fibril forming conditions, αS undergoes multiple aggregation pathways. (A) Schematic of the multiple assembly pathways and products formed by αS. In vitro αS monomers can self-assemble in the same conditions into multiple species of oligomers or into amyloid fibrils, creating a complex heterogeneous environment. Stable oligomers may be formed off-pathway and do not proceed to amyloid formation. Fibril formation is accelerated through fibril seeding processes, such as templated elongation and secondary nucleation. Here, we investigate αS monomer interactions with off-pathway PFOs or PFFs (magenta, dashed arrows) and the impact of coexisting off-pathway PFOs on kinetics of fibril seeding. The monomer ensemble is based on PED00024 (73). Stable oligomers formed in fibril formation conditions have been shown to have a disordered C-terminal cloud (∼55 residues, red) that surrounds the core consisting of the N-terminal and NAC regions (14). Mature amyloid fibrils also have a surface of disordered N- (blue) and C-termini (red) that flank a rigid core [model adapted from PDB: 6h6b (20)]. (B) In an in vitro ThT fibril seeding assay at 37 °C, Ac-αS PFFs induce the formation of new amyloid fibrils (black 40 µM monomer, 1 µM fibril seeds). However, big PFOs do not have the ability to induce formation of new amyloid fibrils (blue 40 µM monomer, 1 µM big PFOs). (C) A TEM image of isolated big PFOs displays their worm-like morphology.It has been observed previously that the rigid amyloid core can act as a template to seed fibril formation (17, 18), yet the role of intrinsically disordered flanking regions in fibril seeding is not well understood. Structural models derived from solid-state NMR and cryo-electron microscopy (EM) have revealed that αS fibrils consist of two protofilaments with a rigid “Greek key” core flanked by intrinsically disordered N- and C-termini (∼40 residues each) (19–23) (Fig. 1A). Current inhibition methods primarily target the rigid core. These include using chaperones to block fibril ends or fibril surfaces (9), treating with natural products that promote fibril clustering to reduce fibril fragmentation and hide binding sites for monomer addition (24, 25), and creating high affinity interactions with amyloidogenic monomers to sequester monomers from self-assembly (26). Recently, it has become increasingly more important to understand the role of the intrinsically disordered regions (IDRs) in fibril growth in order to determine their potential for amyloid inhibition. These IDRs are a substantial fraction of the fibril structure and make up a “fuzzy coat” that surrounds the rigid core (19–21). The flexible flanking regions in several amyloid proteins have been found to be highly involved in biological interactions, including binding to receptors, chaperones, membranes, or RNA (27). In αS, modifying the “fuzzy coat” through pathologically relevant N- and C-terminal truncations was shown to modulate the aggregation and seeding propensity of αS fibrils and influence the resulting fibril morphologies (28–30). This suggests that the terminal IDRs are also actively involved in the αS aggregation mechanism. In order to more fully understand the seeding process, it is critical to identify the monomer–fibril interactions and whether they involve the rigid core or the flanking IDRs.The weak, transient nature of the monomer–fibril interactions that occur during fibril seeding make it a challenge to identify the key residues involved. Solution NMR is unique in its ability to detect perturbations to soluble proteins at atomic-level resolution. Sophisticated solution NMR experiments, dark-state exchange saturation transfer (DEST), have been designed to probe the exchange of NMR-visible, free monomers with the surface of large, NMR-invisible complexes, such as amyloid fibrils, in residue-specific detail not accessible by other techniques. These powerful NMR methods were introduced by probing the interactions between NMR-visible amyloid-β (Aβ) monomers with large, NMR-invisible Aβ protofibrils implicated in amyloid formation (31). More recently, studies have used DEST to determine site specific interactions of IDPs with membranes (32), lipopolysaccharides (33), unilamellar vesicles (34), and in self-assembly (35, 36). Our laboratory has recently used ¹⁵N-DEST to determine the interaction interfaces on immunoglobulin protein β₂-microglobulin (β₂m) for collagen I, which facilitates β₂m amyloid formation (37).Here, we use NMR to characterize monomer–fibril and monomer–oligomer interactions that occur under conditions promoting templated elongation in order to gain a molecular understanding of the interaction between the N-terminally acetylated-αS (Ac-αS) monomer and these complex aggregates. Through relaxation-based NMR experiments, we find that Ac-αS preformed fibrils (PFFs) and off-pathway preformed oligomers (PFOs) are in a dynamic equilibrium with NMR-visible monomers. ¹⁵N-DEST experiments and ¹⁵N-transverse relaxation measurements reveal that the N-terminal 11 residues of Ac-αS monomers interact with PFFs and stable off-pathway PFOs, suggesting that the binding interface of the monomer is similar for both fibrils and oligomers. When added to a monomer solution prepared under templated elongation conditions, these same PFOs significantly delay fibril formation in a concentration-dependent manner and can act as auto-inhibitors of amyloid formation when coexisting with monomers and PFFs. Our results suggest that delays in fibril formation by PFOs may be explained by competing monomer N-terminal interactions between PFFs and PFOs through their common binding modes. Identification of this shared N-terminal binding site on Ac-αS monomers for fibrils and off-pathway oligomers, taken together with previous data, leads us to propose that the N terminus of the intrinsically disordered monomer interacts with the intrinsically disordered terminal flanking regions (IDR) of the fibrils and oligomers, a shared feature of these aggregate structures. These IDP–IDR interactions may play a critical role in recruiting monomer to the fibril under conditions that promote templated elongation. While the structured fibril core is known to be critical to the templated assembly of amyloid fibrils, it is important to consider the role of the N- and C-terminal fibril IDRs in fibril seeding and assembly and their potential as targets against amyloid propagation.     

Atomic-scale evidence for highly selective electrocatalytic N−N coupling on metallic MoS2

Molybdenum sulfide (MoS₂) is the most widely studied transition-metal dichalcogenide (TMDs) and phase engineering can markedly improve its electrocatalytic activity. However, the selectivity toward desired products remains poorly explored, limiting its application in complex chemical reactions. Here we report how phase engineering of MoS₂ significantly improves the selectivity for nitrite reduction to nitrous oxide, a critical process in biological denitrification, using continuous-wave and pulsed electron paramagnetic resonance spectroscopy. We reveal that metallic 1T-MoS₂ has a protonation site with a pK_a of ∼5.5, where the proton is located ∼3.26 Å from redox-active Mo site. This protonation site is unique to 1T-MoS₂ and induces sequential proton−electron transfer which inhibits ammonium formation while promoting nitrous oxide production, as confirmed by the pH-dependent selectivity and deuterium kinetic isotope effect. This is atomic-scale evidence of phase-dependent selectivity on MoS₂, expanding the application of TMDs to selective electrocatalysis.

Transition-metal dichalcogenides (TMDs) have gained considerable attention in recent years due to their variable crystal phases, which allow for precise tuning of their electronic, optical, magnetic, and catalytic properties (1, 2). For example, molybdenum sulfide (MoS₂), which is one of the most extensively studied TMDs, exists as different polymorphs depending on the orientation of sulfur atoms around the molybdenum center. In octahedral coordination (1T phase), MoS₂ exhibits metallic behavior, whereas the material acts as a semiconductor in trigonal prismatic coordination (2H phase) (3–6). In addition to higher conductivity, 1T-MoS₂ has enlarged layer spacing and more electrochemical active sites (7, 8), making it a promising next-generation material for batteries (9, 10), memristors (11, 12), capacitors (13, 14), and numerous other energy-related applications (15–17).In the field of electrocatalysis, phase engineering has mainly been used to enhance catalytic activity. For instance, exchanging 2H-MoS₂ for 1T-MoS₂ results in a marked increase toward the hydrogen evolution reaction (18, 19). Considering the advantage of TMDs being able to control the atomic-scale structure, phase engineering may also open possibilities to control the selectivity of multielectron/proton reactions with multiple possible products, such as CO₂ reduction (20–23), denitrification (NO₃⁻/NO₂⁻ reduction) (24–26), and the electrosynthesis of functional molecules (27–30). Selectivity is a critical requirement for cascade catalysis, one-pot reaction systems, and multistep catalytic processes, and strategies to guide the complex chemical reaction network toward the desired end product are necessary (31, 32). However, to the best of our knowledge, no studies have attempted to exploit the advantages of phase-engineered materials for selective electrocatalysis.One effective approach to explore phase-engineered MoS₂ for selectivity control is to utilize the newly proposed concept of sequential proton−electron transfer (SPET) (off-diagonal pathways, Fig. 1A) (33, 34). In contrast to the extensively studied concerted proton−electron transfer (CPET) pathway, the energy landscape of sequential (decoupled) proton−electron transfer (SPET) pathways is pH-dependent (Fig. 1B). This leads to pH-dependent reaction rates (Fig. 1C), where the maximum reaction rate can be obtained at a pH close to the pK_a of the reaction intermediate (33, 34). This was recently observed experimentally for nitrite reduction to dinitrogen – an artificial analog of biological denitrification – on partially oxygenated molybdenum sulfide (oxo-MoS_x), and the record high selectivity toward dinitrogen was achieved by simple pH optimization (35). In contrast, this pH dependence was absent in the case of crystalline 2H-MoS₂, demonstrating that the SPET pathway is a unique property of oxo-MoS_x and is therefore probably phase-dependent. However, the origin of the SPET behavior on this material remains unclear. Therefore, elucidating the mechanism at the atomic level would help rationalize the relationship between selectivity and crystal phases, thus providing significant insight into the newly proposed SPET mechanism (33, 34) to enhance the selectivity of multistep electrochemical processes.Open in a separate windowFig. 1.Selectivity control of MoS₂ based on SPET theory. (A) Diagram showing the possible pathways for proton−electron transfer on MoS₂. In the blue pathway (CPET), protons and electrons are transferred in a single elementary step. In contrast, stepwise pathways (SPET) generate an intermediate whose charge depends on whether the electron or proton transfers first (red and black pathways, respectively). (B) Diagram showing the energetic landscape of SPET. The landscape depends on the relationship between the pK_a of the reaction intermediate and the solution pH. (C) Influence of pH on reaction selectivity. The rates of SPET reactions (red lines) show a pH dependence with a maximum corresponding to the pK_a of the intermediate. Therefore, the relative rate of one reaction over another can be tuned by changing the pH. In contrast, the rate of CPET reactions are pH-independent, and therefore, their relative rates are also constant with respect to pH.Here, we identified the atomic-scale origin of SPET-driven selectivity on MoS₂ using continuous-wave electron paramagnetic resonance (CW-EPR), Raman, and pulsed ¹H/²H electron−nuclear double-resonance (ENDOR) spectroscopy. Specifically, a proton located at the first coordination sphere (∼3.26 Å) of a redox-active Mo center was found to have a pK_a value matching that involved in the pH-dependent electrocatalytic selectivity and H/D kinetic isotope effect (KIE). The observed pH-dependent behavior is specific to 1T-MoS₂, as oxo-MoS_x was assigned to the 1T phase using high-resolution transmission electron microscopy (HRTEM), Raman- and X-ray photoelectron spectroscopy (XPS). These results not only provide atomic-scale evidence of SPET in heterogeneous catalysis, but also demonstrate how the phase engineering of TMDs can be used to enhance their electrocatalytic selectivity.     

Capillary equilibrium of bubbles in porous media

In geologic, biologic, and engineering porous media, bubbles (or droplets, ganglia) emerge in the aftermath of flow, phase change, or chemical reactions, where capillary equilibrium of bubbles significantly impacts the hydraulic, transport, and reactive processes. There has previously been great progress in general understanding of capillarity in porous media, but specific investigation into bubbles is lacking. Here, we propose a conceptual model of a bubble’s capillary equilibrium associated with free energy inside a porous medium. We quantify the multistability and hysteretic behaviors of a bubble induced by multiple state variables and study the impacts of pore geometry and wettability. Surprisingly, our model provides a compact explanation of counterintuitive observations that bubble populations within porous media can be thermodynamically stable despite their large specific area by analyzing the relationship between free energy and bubble volume. This work provides a perspective for understanding dispersed fluids in porous media that is relevant to CO₂ sequestration, petroleum recovery, and fuel cells, among other applications.

Bubbles are generated, trapped, and mobilized within porous media as a consequence of incomplete fluid–fluid displacements (1, 2), phase changes (3, 4), chemical and biochemical reactions (5, 6), or injection of emulsified fluids and foams (7, 8). Compared to continuously connected phases, the behavior of dispersed bubbles, or ganglia, are far less understood. In particular, the thermodynamic stability of bubbles, despite their large specific surface area, remains a puzzle. The difficulty comes from the fact that each bubble can attain a volume (V), topology, and capillary pressure (P_c) that is distinct from other bubbles in the medium (9). The variability poses challenges to understanding the transport and trapping mechanisms of bubbles in geologic CO₂ sequestration (10, 11), hydrocarbon recovery (12, 13), fuel cell water management (14, 15), and vadose zone oxygen supply (16, 17).The dominant factor controlling a bubble’s behavior in a porous medium is capillarity, which is typically much larger than either viscous, gravitational, or inertial forces (18, 19). Capillary pressure, P_c, allows a closure relationship for two-phase Darcy Eqs. (20–22) and influences thermodynamic properties like phase partition (23). Capillary pressure is derived from the Young–Laplace equation P_c = γκ, where γ is the interfacial tension and κ is the surface curvature. In an open space without obstacles, a bubble spontaneously evolves into a sphere to minimize its total interfacial energy. Thus, P_c is a continuous and monotonically decreasing function of V (Fig. 1A). However, in a porous medium, bubble’s P_c–V relation is more complicated due to the geometric confinement imposed by the porous structure and topological evolution (24). A bubble can no longer remain spherical as it grows in size but must conform to the geometry of the pore(s) it occupies. Therefore, a bubble’s P_c is a function of not only its volume and interfacial tension but also its topology as dictated by the confining porous medium, as confirmed by recent laboratory experiments and numerical simulations (25–29). The mere presence of confinement therefore engenders a host of phenomena that would otherwise be absent, such as capillary trapping (30, 31), anticoarsening of bubble populations (32, 33), and complex ganglion dynamics (11, 18). Furthermore, theoretical studies in mathematical topology (28, 34, 35) prove that immiscible fluids can be fully characterized by d+1 Minkowski functionals, where d is the problem dimension. Such characterizations remove the path-dependent (or hysteretic) behavior common to these systems (34, 35).Open in a separate windowFig. 1.(A) Spherical bubbles inside a bulk fluid. (B) Micromodel observations show that bubbles are nonspherical in porous media and may occupy multiple pores. This image is from SI Appendix, Movie S1. (C) A 2D porous medium comprised of an ordered array of identical circular grains. A bubble occupying multiple pores including a zoom-in to a portion of it. (D) Illustration of the full state. (E) Illustration of the critical state. (F) Decomposition of a bubble into four distinct parts: minor arc menisci shown by dark blue cap-shaped regions, throats shown by light blue diamond-shaped regions, inner bulk bodies shown by red star-shaped regions, and major arc menisci shown by dark green cap-shaped regions.Recent developments in microfluidics and micro computed tomography imaging allow detailed pore-scale visualizations of fluids inside porous media, including the morphology of bubbles and ganglia (25, 36–39). Garing et al. (25) experimentally measured the equilibrium capillary pressure of trapped air bubbles inside sandstone and bead-pack samples. They found that, unlike bubbles within a bulk fluid, the P_c of trapped bubbles shows no clear dependence on V and seems to fall within a bounded interval, except for vanishingly small V. Xu et al. (40) proposed an empirical correlation for the P_c trapped bubbles based on microfluidic observations. In this correlation, as V increases, P_c decreases until a minimum is reached and then increases linearly. In the first stage, the bubble is unconfined, whereas in the second, it is reshaped by the surrounding solid walls. The proposed correlation, however, is only valid for bubbles in a single pore and not bubbles that span multiple pores. The latter seems to be rather common in nature as evidenced by recent direct observations (Fig. 1B) (2, 25).Here, we propose a simple conceptual model to describe the equilibrium states of a bubble with arbitrary size trapped inside a porous medium. The model accounts for the bubble’s morphology, the geometry of the solid matrix, and the wettability between the two. We derive all metastable configurations of the bubble analytically and highlight the thermodynamic states the bubble assumes when it is static, growing, or shrinking. We also show that the relationship between surface free energy (F) and volume (V) of large bubbles is approximately linear, which explains the previously counterintuitive observation that such bubbles are thermodynamically stable despite having large surface areas. Our work provides a step toward understanding the capillary state, stability, and evolution of dispersed immiscible fluids in porous media.     

Structural and functional characterization of the pore-forming domain of pinholin S2168

Pinholin S²¹68 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state ¹⁵N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (S_mol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G₄₀xxxS₄₄xxxG₄₈) together with a patch of H-bonding residues (T₅₁, T₅₄, N₅₅) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Upon infecting a host, bacteriophages release their newly produced offspring into the environment by lysis of the host cell. The infection cycle of double-stranded DNA bacteriophages that infect gram-negative bacteria is regulated by small viral membrane proteins, so-called holins (1–3). These holins accumulate in an inactive form in the hosts’ cytoplasmic membrane during phage morphogenesis (3). At an allele-specific time, the holins start to form big membrane lesions. Through these holes the muralytic endolysins escape from the cytoplasm and start to degrade the peptidoglycane layer (4, 5). Host lysis is then completed by the Rz–Rz1 spanin complex that disintegrates the outer membrane (6). In addition to this canonical holin endolysin system, a second class of holins, referred to as pinholins, has been described (3, 7). This class is represented by pinholin S²¹68 of lambdoid phage φ21 (7, 8). In contrast to canonical holins, for example those encoded by phage λ, the holes formed by pinholins are much smaller (“pinholes”) and do not allow the passage of proteins (7, 9).The pinholin S²¹68 protein is encoded by gene S²¹ of the lysis cassette, which possesses a dual start motif to allow expression of an antipinholin as well. This antipinholin protein has three additional N-terminal amino acids (M₁-K₂-S₃) and serves as a specific inhibitor of pinholin (Fig. 1A) (8, 10). The (anti)pinholin protein structure consists of two transmembrane domains (TMDs) and an unstructured C terminus (8). It is postulated that TMD1 possesses regulatory functions, while TMD2 is responsible for the actual pinhole formation (8, 11–13). During phage morphogenesis, (anti)pinholin initially accumulates as inactive heterodimers in the cytoplasmic membrane. For pinhole formation, TMD1 has to flip out of the membrane to allow TMD2–TMD2 interactions that build up the pinhole (Fig. 1B) (8, 14). The presence of antipinholin reduces the pinholin activity, because the additional charge on the N terminus delays the flipping of TMD1 (8, 11). Previous cross-linking and modeling studies by Pang et al. (9) have suggested that the pinholes are composed of seven pinholin S²¹68 monomers. These heptamers are supposed to form small holes with an inner diameter of around 1.5 nm, which lead to a collapse of the proton gradient across the membrane (9).Open in a separate windowFig. 1.Pinholin S²¹68. (A) Primary structure of the (anti)pinholin. The dual start motif of gene S²¹ of phage φ21 allows the expression of both antipinholin S²¹71 and pinholin S²¹68. The postulated TMDs are indicated, and our synthetic TMD2 fragment (W₃₆-E₆₂) is underlined. (B) In its inactive state, both helices of pinholin S²¹68 are supposed to be aligned in a transmembrane state (Left). Above a certain threshold concentration, TMD1 is supposed to flip out of the membrane into a surface bound state (Right) (14). This triggering allows further self-assembly through TMD2–TMD2 interactions and leads to the formation of a small pinhole pore by a putative heptamer (9, 13).The machinery also contains associated endolysins carrying an N-terminal secretory signal, called the signal-anchor release (SAR) domain. In their membrane-tethered state, the SAR-endolysins are enzymatically inactive, which prevents premature lysis (15, 16). This inactive state is highly dependent on the presence of an intact membrane potential (7, 17). After disruption of this potential by the formation of pinholes, the SAR-endolysins are released from the membrane, and become refolded and activated during this process (7, 16).Drew et al. (18) demonstrated recently that the wild-type protein is predominately α-helical (∼83%) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine; di-14:0-PC) vesicles. Ahammad et al. (14) from the same group used electron paramagnetic resonance (EPR) experiments on synthetic reconstituted pinholin to show that TMD1 is partly externalized from the lipid, whereas TMD2 is stably membrane inserted. In the present study, we aimed to determine the detailed membrane orientation of the postulated pore-forming TMD2 fragment and examine its putative role as the basic homo-/hetero-/oligomeric assembly unit. We used synchrotron radiation-based circular dichroism spectroscopy (SRCD) for quantitative secondary structure determination, and oriented CD (SROCD) for qualitative orientational analysis. Accurate parameters on the helix alignment were obtained by solid-state NMR spectroscopy (ssNMR) on nonperturbing selective ¹⁵N-isotope labels, which yielded a detailed picture of the TMD2 as it is positioned in the membrane. Using a vesicle leakage assay based on ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt)/DPX (p-xylene-bis-pyridinium bromide), we proved that TMD2 is indeed the pore-forming domain and could estimate the size of the pinhole from the leakage of fluorescein-isothiocyanate-dextrans (FITC-dextrans, FDs). Based on these experimental data, we have constructed putative models for the pinholin in all three stages of its functional cycle.