Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K⁺ channel α-subunits to form I_Kslow (I_Ks) channels in the heart and ear. The number of E1 subunits in I_Ks channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface I_Ks channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, I_Ks channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce I_Ks channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.Voltage-gated potassium (K_V) channels include four α-subunits that form a single, central ion conduction pathway with four peripheral voltage sensors (1–3). Incorporation of accessory β-subunits modifies the function of K_V channels to suit the diverse requirements of different tissues. KCNE genes encode minK-related peptides (MiRPs) (4–6), β-subunits with a single transmembrane span that assemble with a wide array of K_V α-subunits (7, 8) to control surface expression, voltage dependence, and kinetics of gating transitions, unitary conductance, ion selectivity, and pharmacology of the resultant channel complexes (4, 9–15). I_Kslow (I_Ks) channels in the heart and inner ear are formed by the α-subunit encoded by KCNQ1 (called Q1, K_VLQT1, K_V7.1, or KCNQ1) and the β-subunit encoded by KCNE1 (called E1, mink, or KCNE1) (16, 17). Inherited mutations in Q1 and E1 are associated with cardiac arrhythmia and deafness.The number of E1 subunits in I_Ks channels has been a longstanding matter of disagreement. We first argued for two E1 subunits per channel based on the suppression of current by an E1 mutant (18). Subsequently, we reached the same conclusion by determining the total number of channels using radiolabeled charybdotoxin (CTX), a scorpion toxin that blocks channels when one molecule binds in the external conduction pore vestibule, and an antibody-based luminescence assay to tally E1 subunits (19). Morin and Kobertz (20) used iterative chemical linkage between CTX in the pore and E1, and they also assigned two accessory subunits to >95% of I_Ks channels without gathering evidence for variation in subunit valence. Furthermore, when we formed I_Ks channels from separate E1 and Q1 subunits and compared them with channels enforced via genetic encoding to contain two or four E1 subunits (19), we observed the natural I_Ks channels to have the same gating attributes, small-molecule pharmacology, and CTX on and off rates (a reflection of pore vestibule structure) as channels encoded with two E1 subunits but not those with four. These findings support the conclusion that two E1 subunits are necessary, sufficient, and the normal number in I_Ks channels.In contrast, others have argued that I_Ks channels have variable stoichiometry with one to four E1 subunits, or even more (21–24). Recently, Nakajo et al. (25) applied single-particle spectroscopy to the question; this powerful “gold-standard” tool has been a valuable strategy to assess the subunit composition of ion channels (26–28) and should be expected to improve on prior investigations conducted on populations of I_Ks channels and subject, therefore, to the simplifying assumptions that attend macroscopic studies (29). Nakajo et al. (25) reported a variable number of E1 subunits, from one to four, in I_Ks channels studied in Xenopus laevis oocytes. The impact of this result has been striking because it has engendered new models of cardiac physiology, altered models of I_Ks channel biosynthesis and function, stimulated the use of transgenic animals artificially enforced to express I_Ks channels with four E1 subunits (by expression of a fused E1–Q1 subunit), and prompted cardiac drug design based on the assumption that I_Ks channels can form with one E1 subunit (23, 30–32).We were concerned that the conclusions of Nakajo et al. (25) were in error because they appraised only a limited fraction of particles that were immobile in the oocyte membrane; counted E1 and Q1 asynchronously rather than simultaneously (increasing the risk that particles moved into or out of the field of view); and studied Q1 and E1 appended not only with the fluorescent proteins (FP) required to count subunits by photobleaching but also with a common trafficking motif that suppressed channel mobility by interacting with an overexpressed anchoring protein, thereby risking nonnatural aggregation of subunits.Here, to resolve mobility problems and obviate the need for modification of subunits with targeting motifs, we describe and perform single-fluorescent-particle photobleaching at the surface of live mammalian cells, demonstrating three spectroscopic counting approaches: standard, asynchronous subunit counting; simultaneous, two-color subunit counting; and toxin-directed, simultaneous, two-color photobleaching. To analyze the data, we use two statistical approaches—one to assess the degree of colocalization of objects in dual-color images (33) and the other to infer stoichiometry from single-molecule photobleaching (34). These methods also allow determination of the surface density of assemblies of defined subunit composition and are therefore useful to assess the formation and life cycle of membrane protein complexes.We report that single I_Ks channels at the surface of mammalian cells contain two E1 subunits—no more and no less. This finding refutes the single-particle studies of Nakajo et al. (25) in oocytes and macroscopic studies (21–24, 30–32), arguing that forcing cells to express excess E1 produces I_Ks channels containing more than two E1 subunits and that low levels of E1 yields I_Ks channels with less than two E1 subunits. Not once did we observe an I_Ks channel with three or four E1 subunits. Moreover, simultaneous, two-color subunit counting revealed that low amounts of E1 relative to Q1 [ratios like those reported in human cardiac ventricle (35, 36)] produced two types of channels on the cell surface: I_Ks channels (with two E1 subunits) and Q1 channels (with no E1 subunits). Finally, E1 was shown to increase in I_Ks channel surface expression threefold, as we predicted based on assessment of I_Ks channel unitary conductance (11), whereas few E1 subunits were on the surface outside of I_Ks channels, even when E1 was expressed alone. This finding indicates that E1 does not travel to the surface readily on its own, that two E1 subunits facilitate I_Ks channel trafficking to the surface (or enhance surface residence time compared with Q1 channels), and that I_Ks channels with only one E1 subunit do not form, do not reach the surface, or are rapidly recycled.     

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

H⁺-transporting F₁F₀ ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F₀ and F₁. H⁺ transport at the subunit a–c interface in transmembranous F₀ drives rotation of a cylindrical c₁₀ oligomer within the membrane, which is coupled to rotation of subunit γ within the α₃β₃ sector of F₁ to mechanically drive ATP synthesis. F₁F₀ functions in a reversible manner, with ATP hydrolysis driving H⁺ transport. ATP-driven H⁺ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag⁺ and/or Cd⁺² with the substituted sulfhydryl groups. The H⁺ transport pathway mapped via these Ag⁺(Cd⁺²)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H⁺ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd⁺² directly inhibits passive H⁺ transport mediated by F₀ reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H⁺ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H⁺ transport likely interact in a single structural domain, which then functions in gating H⁺ release to the cytoplasm.The F₁F₀-ATP synthase of oxidative phosphorylation uses the energy of a transmembrane electrochemical gradient of H⁺ or Na⁺ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F₁ and F₀ sectors of the enzyme (1). H⁺ transport through the transmembrane F₀ sector is coupled to ATP synthesis or hydrolysis in the F₁ sector at the surface of the membrane. Homologous ATP synthases are found in mitochondria, chloroplasts, and many bacteria. In Escherichia coli and other eubacteria, F₁ consists of five subunits in an α₃β₃γδε stoichiometry. F₀ is composed of three subunits in a likely ratio of a₁b₂c₁₀ in E. coli and Bacillus PS3 (2, 3) or a₁b₂c₁₁ in the Na⁺ translocating Ilyobacter tartaricus ATP synthase (1, 4) and may contain as many as 15 c subunits in other bacterial species (5). Subunit c spans the membrane as a hairpin of two α-helices, with the first transmembrane helix (TMH) on the inside and the second TMH on the outside of the c ring (1, 4). The binding of Na⁺ or H⁺ occurs at an essential, membrane-embedded Glu or Asp on cTMH2. High-resolution X-ray structures of both Na⁺- and H⁺-binding c-rings have revealed the details and variations in the cation binding sites (4–8). In the H⁺-translocating E. coli enzyme, Asp-61 at the center of cTMH2 is thought to undergo protonation and deprotonation, as each subunit of the c ring moves past the stationary subunit a. In the functioning enzyme, the rotation of the c ring is thought to be driven by H⁺ transport at the subunit a/c interface. Subunit γ physically binds to the cytoplasmic surface of the c-ring, which results in the coupling of c-ring rotation with rotation of subunit γ within the α₃β₃ hexamer of F₁ to mechanically drive ATP synthesis (1).E. coli subunit a folds in the membrane with five TMHs and is thought to provide aqueous access channels to the H⁺-binding cAsp-61 residue (9, 10). Interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation–protonation cycle of cAsp-61 (1, 11, 12). At this time, very limited biophysical or crystallographic information is available on the 3D arrangement of the TMHs in subunit a. TMHs 2–5 of subunit a pack in a four-helix bundle, which was initially defined by cross-linking (13), but now, such a bundle, packing at the periphery of the c-ring, has been viewed directly by high-resolution cryoelectron microscopy in the I. tartaricus enzyme (14). Previously published cross-linking experiments support the identification of aTMH4 and aTMH5 packing at the periphery of the c-ring and the identification of aTHM2 and aTMH3 as the other components of the four-helix bundle seen in these images (13, 15, 16). More recently, published cross-linking experiments identify the N-terminal α-helices of two b subunits, one of which packs at one surface of aTMH2 with close enough proximity to the c-ring to permit cross-linking (17). The other subunit b N-terminal helix packs on the opposite peripheral surface of aTMH2 in a position where it can also be cross-linked to aTMH3 (17). The last helix density shown in Hakulinen and colleagues (14) packs at the periphery of the c-ring next to aTMH5 and is very likely to be aTMH1.The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag⁺ and other thiolate-reactive agents (18–20). Two regions of aqueous access were found with distinctly different properties. One region in TMH4, extending from Asn-214 and Arg-210 at the center of the membrane to the cytoplasmic surface, contains Cys substitutions that are sensitive to inhibition by both N-ethylmaleimide (NEM) and Ag⁺ (18–20; Fig. 1). These NEM- and Ag⁺-sensitive residues in TMH4 pack at or near the peripheral face and cytoplasmic side of the modeled four-helix bundle (11, 13). A second set of Ag⁺-sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18, 19), and Ag⁺-sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19, 20). The Ag⁺-sensitive substitutions on the periplasmic side of TMHs 2–5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11, 13, 19, 20). We have proposed that the movement of H⁺ from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a–c subunit interface (16, 21–24). This gating is thought to be coupled with ionization of a protonated cAsp61 in the adjacent subunit of the c-ring and with release of the H⁺ into the cytoplasmic half-channel at the subunit a–c interface. The route of aqueous access to the cytoplasmic side of the c subunit packing at the a–c interface has also been mapped by the chemical probing of Cys substitutions and, more recently, by molecular dynamics simulations (22, 25, 26).Open in a separate windowFig. 1.The predicted topology of subunit a in the E. coli inner membrane. The location of the most Ag⁺-sensitive Cys substitutions are highlighted in red (>85% inhibition) or orange (66–85% inhibition). The five proposed TMHs are shown in boxes, each with a span of 21 amino acids, which is the minimum length required to span the hydrophobic core of a lipid bilayer. The α-helical segments shown in loops 1–2 and 3–4 are consistent with the predictions of TALOS, based on backbone chemical shifts seen by NMR (29). Others have also predicted extensive α-helical regions in these loops (12, 30), but the possible positions remain largely speculative. aArg210 is highlighted in green. Figure is modified from those shown previously (21, 23, 24, 27).We have also reported Ag⁺-sensitive Cys substitutions in two cytoplasmic loops of subunit a (27) and, more recently, in the cytoplasmic loop of subunit c (28). The mechanism by which Ag⁺ inhibited F₁F₀-mediated H⁺ transport was uncertain. Several of these substitutions were also sensitive to inhibition by Cd⁺², and these substitutions provided a means of testing whether Cd⁺² directly inhibits passive H⁺ transport through F₀ (28). In the case of two subunit c loop substitutions, Cd⁺² was shown to directly inhibit passive F₀-mediated transport activity. In this study, we have extended the survey to Cd⁺²-sensitive Cys substitutions in cytoplasmic loops of subunit a. We report four loop substitutions in which Cd⁺² inhibits passive F₀-mediated H⁺ transport. Further, in two cases, we show cross-linking between pairs of Cys substitutions, which lie in subunits a and c, respectively, and which individually mediate passive H⁺ transport activity. These results suggest that the a and c loops, which gate H⁺ release to the cytoplasm, fold into a single domain at the surface of F₀.     

Structural basis for high-voltage activation and subtype-specific inhibition of human Nav1.8

The dorsal root ganglia–localized voltage-gated sodium (Na_v) channel Na_v1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Na_v1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Na_v1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensing domain (VSD_I) displays three different conformations. Structure-guided mutagenesis identified the extracellular interface between VSD_I and the pore domain (PD) to be a determinant for the high-voltage dependence of activation. A-803467 was clearly resolved in the central cavity of the PD, clenching S6_IV. Our structure-guided functional characterizations show that two nonligand binding residues, Thr397 on S6_I and Gly1406 on S6_III, allosterically modulate the channel’s sensitivity to A-803467. Comparison of available structures of human Na_v channels suggests the extracellular loop region to be a potential site for developing subtype-specific pore-blocking biologics.

Voltage-gated sodium (Na_v) channels govern membrane excitability in neurons and muscles (1, 2). Despite high-degree sequence and architectural similarity, different subtypes of Na_v channels have specific tissue distributions and distinct voltage dependence and kinetics for activation, inactivation, and recovery (3). Among the nine mammalian Na_v channels (SI Appendix, Fig. S1), Na_v1.8, a tetrodotoxin (TTX)-resistant subtype encoded by SCN10A, is primarily expressed in the sensory neurons, exemplified by the dorsal root ganglia (DRG) neurons (4–6). Compared with other Na_v subtypes, Na_v1.8 has several unique biophysical properties, such as activation at more depolarized voltage and slower inactivation with persistent current, which enable the hyperexcitability of the DRG neurons (4, 5, 7–11).Na_v1.8 functions in pain sensation (12–16). Proexcitatory mutations of Na_v1.8 were identified in patients with painful small fiber neuropathy (17–19). On the other hand, a natural variant, A1073V, that shifts the voltage dependence of activation to more depolarized direction appeared to ameliorate pain symptoms (20). Specific inhibition of the peripheral Na_v1.8 thus represents a potential strategy for developing nonaddictive pain killers (21, 22).Several Na_v1.8-selective blockers, such as VX-150 and PF-06305591, had been tested in clinical trials. However, most of the drug candidates failed to meet the endpoint(s) of phase II trials for various reasons, such as unsatisfactory efficacy or selectivity (21, 23–26). Structures of Na_v1.8 bound to lead compounds will shed light on drug optimization for improving potency and selectivity. We focused on A-803467, a Na_v1.8-selective blocker, for structural analysis. A-803467 was shown to inhibit Na_v1.8 in both the resting state and inactivated state. Despite a wide range of concentration that inhibits response by 50% (IC₅₀) from several nanomolar to 1 μM measured by different groups, A-803467 consistently shows a higher affinity for the inactivated channel (27–31).In this study, we report the structures of full-length human Na_v1.8 alone and bound to A-803467. The first voltage-sensing domain (VSD_I) was resolved in multiple conformations. Based on the structural and electrophysiological characterizations, we attempt to address two questions: What underlies the high-voltage activation of Na_v1.8, and what determines the subtype specificity of A-803467?     

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Excitation-evoked Ca²⁺ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca²⁺ entry are thought to be functions of voltage-gated calcium (Ca_V) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of Ca_V1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in Ca_V1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.Voltage-gated Ca²⁺ (Ca_V) channels respond to membrane depolarization by catalyzing Ca²⁺ influx. Ca_V-mediated elevation of intracellular [Ca²⁺] regulates such critical physiological functions as neurotransmitter and hormone release, axonal outgrowth, muscle contraction, and gene expression (1). Their relevance to human physiology is evident from the broad phenotypic consequences of Ca_V channelopathies (2). The voltage dependence of Ca_V-driven Ca²⁺ entry relies on the modular organization of the channel-forming α₁ subunit (Fig. 1), which consists of four repeated motifs (I–IV), each comprising six membrane-spanning helical segments (S1–S6) (Fig. 1A). Segments S1–S4 form a voltage-sensing domain (VSD), whereas segments S5 and S6 contribute to the Ca²⁺-conductive pore (1). The VSDs surround the central pore (Fig. 1B). VSDs are structurally and functionally conserved modules (3–5) capable of transducing a change in the cell membrane electrical potential into a change of ion-specific permeability or enzyme activity. VSDs sense depolarization by virtue of a signature motif of positively charged Arg or Lys at every third position of helix S4 (Fig. 1D), which rearranges in response to depolarization (4, 6–10). In contrast to voltage-gated K⁺ (K_V) channels but similar to pseudotetrameric voltage-gated Na⁺ (Na_V) channels, the amino acid sequences encoding each VSD have evolved independently (Fig. 1D). In addition to their distinct primary structure, the four Ca_V VSDs may also gain distinct functional properties from the asymmetrical association of auxiliary subunits, such as β, α₂δ, and calmodulin (1, 11–16) (Fig. 1C). The structural divergence among VSD-driven channels was foreseen by the classical Hodgkin–Huxley model (17), in which four independent “gating particles” control the opening of homotetrameric K_V channels and only three seem sufficient to open Na_V channels. An early study by Kostyuk et al. (18) suggested that only two gating particles are coupled to Ca_V channel opening. We recognize today that gating particles correspond to VSDs, and in Na_V channels, VSDs I–III control Na⁺ influx, whereas VSD IV is associated with fast inactivation (19–21).Open in a separate windowFig. 1.Ca_V membrane topology, putative structure, and S4 helix homology. (A) Ca_V channel-forming α₁ subunits consist of four concatenated repeats, each encompassing one voltage sensor domain (VSD) and a quarter of the central pore domain (PD) (1). Stars indicate the positions of fluorophore labeling. (B) The atomic structure of an Na_V channel (Protein Data Bank ID code 4EKW; top view) (56) shown as a structural representation for the Ca_V α₁ subunit. (C) The α₁ subunit asymmetrically associates with auxiliary β, α₂δ, and calmodulin (CaM) subunits (11–16). (D) Sequence alignment of VSD helix S4 from each of four Ca_V1.2 repeats and the archetypal homotetrameric Shaker K⁺ channel. Conserved, positively charged Arg or Lys is in blue. Residues substituted by Cys for fluorescent labeling are marked: F231 (VSD I), L614 (VSD II), V994 (VSD II), and S1324 (VSD IV).In this study, we used fluorometry to probe the properties of four individual VSDs in a human L-type calcium channel Ca_V1.2, which is a widely expressed regulator of physiological processes, such as cardiac and smooth muscle contractility (22). Although the collective transition of the Ca_V VSDs and the pore has been investigated in studies measuring total charge displacement (gating currents) (23, 24), the activation properties and functional roles of each VSD are unknown. Evidence for the role of each VSD in L-type Ca_V channel operation has been presented from charge neutralization studies, but a clear picture has yet to emerge. Work on a chimeric L-type channel suggests that VSDs I and III drive channel opening (25), whereas other studies on Ca_V1.2 favored the involvement of VSD II over VSD I, with the roles of VSDs III and IV remaining unclear (26, 27).The individual optical reports of four Ca_V1.2 VSDs revealed that each operates with distinct biophysical parameters. We found that VSDs II and III exhibit voltage- and time-dependent characteristics compatible with channel opening and that they can be considered rate-limiting for activation. We compared the voltage and time dependence of the fluorescent signals and ionic currents with the predictions of thermodynamic models relevant to Ca_V domain organization. We found that Ca_V1.2 activation is compatible with a model of allosteric VSD–pore coupling, where VSDs II and III are the primary drivers of channel opening with a smaller contribution by VSD I. We discuss the mechanism of Ca_V1.2 voltage sensitivity, which exhibits similarities to but also clear differences from the related pseudotetrameric Na_V1.4 channels.     

Machine learning–based inverse design for electrochemically controlled microscopic gradients of O2 and H2O2

A fundamental understanding of extracellular microenvironments of O₂ and reactive oxygen species (ROS) such as H₂O₂, ubiquitous in microbiology, demands high-throughput methods of mimicking, controlling, and perturbing gradients of O₂ and H₂O₂ at microscopic scale with high spatiotemporal precision. However, there is a paucity of high-throughput strategies of microenvironment design, and it remains challenging to achieve O₂ and H₂O₂ heterogeneities with microbiologically desirable spatiotemporal resolutions. Here, we report the inverse design, based on machine learning (ML), of electrochemically generated microscopic O₂ and H₂O₂ profiles relevant for microbiology. Microwire arrays with suitably designed electrochemical catalysts enable the independent control of O₂ and H₂O₂ profiles with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s. Neural networks aided by data augmentation inversely design the experimental conditions needed for targeted O₂ and H₂O₂ microenvironments while being two orders of magnitude faster than experimental explorations. Interfacing ML-based inverse design with electrochemically controlled concentration heterogeneity creates a viable fast-response platform toward better understanding the extracellular space with desirable spatiotemporal control.

Ubiquitous spatiotemporal heterogeneity of natural environments fosters the diverse and fascinating biology that our world embraces, and motivates researchers to mimic natural environments with high spatiotemporal resolution (1–5). Given their close relevance in biochemical metabolisms, dioxygen (O₂) and hydrogen peroxide (H₂O₂) as a surrogate of reactive oxygen species (ROS) are two ubiquitous biologically relevant species in extracellular medium (1, 6). Their extracellular spatial and temporal distributions, particularly at the microscopic scale ranging from 1 μm to 100 μm (7–11), are critical for signal transduction, protein expression, biochemical redox balance, and regulation for cellular metabolism with extensive ecological, environmental, and biomedical implications (Fig. 1A) (1, 3, 8–13). A programmable creation of the spatiotemporal concentration profiles of O₂ and H₂O₂ offers the freedom to mimic, control, and perturb the microenvironments of O₂ and H₂O₂ and hence advance our understanding in microbiology.Open in a separate windowFig. 1.AI-based inverse design of electrochemically generated O₂ and H₂O₂ heterogeneities. (A) The ubiquitous spatiotemporal heterogeneities of O₂ and H₂O₂ in microbiology and the challenges posed in this research topic. (B) The combination of electrochemistry and ML-based inverse design offers a viable approach to mimicking and controlling the heterogeneities of O₂ and H₂O₂ in microbiology. O, oxidant; R, reductant; E_appl (t), the time-dependent electrochemical voltages applied on electrodes. (C) The design of the electrochemically active microwire array electrodes for the generation of O₂ and H₂O₂ gradients; 4e⁻ ORR & 2e⁻ ORR, four-electron and two-electron oxygen reduction reaction into H₂O and H₂O₂, respectively. (D and E) The 45°-tilting images of SEM for the representative microwire arrays used for the training of the ML model (D) and the ones inversely designed for targeted O₂ and H₂O₂ gradients (E); k = (P, D, L), the morphological vector that includes the P, D, and L of the synthesized wire arrays in units of micrometers. (Scale bars, 20 μm.)Despite recent progress (14–18), there remain major technical challenges, particularly in the achievable spatiotemporal resolution and high-throughput design of concentration profiles to suit a plethora of scenarios in microbiology. Approaches based on microfluidics and hydrogels have been able to achieve concentration gradients of O₂ and H₂O₂ through the provision of either O₂/H₂O₂ source (14, 19–21), O₂/H₂O₂ scavenging agents (15, 22, 23), or a combination of both (24) across liquid-impermeable barriers such as agar layers or polymeric thin films (25, 26). Yet such approaches, dependent on passive mass transport and diffusion across more than 10² μm, are inherently incapable of achieving spatial features of less than 100 μm and temporal resolution smaller than ∼10¹ s, the prerequisites to investigate microbiology at cluster or single-cell levels (10–12). Moreover, the large variations of extracellular O₂ and H₂O₂ gradients in different microbial systems demand an inverse design strategy, which, with minimal expenditure, quickly programs a desired concentration profile catering to a specific biological scenario (2–5). The current lack of inverse design protocol impedes the adoption of controllable extracellular heterogeneity to mimic and investigate microbial systems that are of environmental, biomedical, and sustainability-related significance.We envision that the integration of electrochemically generated concentration gradients with inverse design based on machine learning (ML) will address the aforementioned challenges (Fig. 1B). Electrochemistry offers a venue for transducing electric signals into microscopic concentration profiles within ∼10⁰ μm to ∼10² μm away from electrodes’ surface, following the specific electrode reaction kinetics and the mass transport governing equations in the liquid phase (27). Proper designs of electrodes’ microscopic spatial arrangement and electrochemical kinetics lead to concentration gradients that are spatiotemporally programmable by time-dependent electric signals of varying voltages (28). Such benefits of electrochemically generated concentration gradients lead us to employ electrochemistry as a tool to spatiotemporally control the concentration profiles in the extracellular medium. In one example, we found that wire arrays electrochemically active toward O₂ reduction create anoxic microenvironment about 20 μm away from the aerobic external bulk environments, modulate the size and extent of O₂ depletion in the anoxic microenvironment by the wire array’s morphology and applied electrochemical potential (E_appl), and hence enable O₂-sensitive rhizobial N₂ fixation in ambient air powered by renewable electricity (29). Moreover, while not reported before as far as we know, electrochemically generated concentration heterogeneity is commensurate with ML-based inverse design (30, 31), thanks to the mathematically well-defined electrochemical processes that can be numerically simulated (32, 33). We recently reported neural networks, trained by numerically simulated data, that explore the influence of electrode geometry on electrochemical N₂ fixation and achieve optimized morphologies of wire array electrodes untenable without such an ML-based strategy (34). An inverse design for the electrochemically generated gradients will quickly program desirable microenvironments of O₂ and ROS with high spatiotemporal resolutions, thanks to the well-reported electrochemical transformation related to O₂ and H₂O₂ with high electrochemical selectivity (35, 36).In this work, we report an inverse design based on neural networks for independent electrochemical creation of O₂ and ROS microscopic gradients that are relevant, and mimic their extracellular heterogeneities in microbial systems. We hypothesize that careful design of electrocatalysis of O₂ reduction reaction (ORR) can either facilitate four-electron ORR on Pt electrocatalyst for a controllable O₂ spatiotemporal profile or promote two-electron ORR on Au electrocatalyst for a programmable generation of H₂O₂ gradient without significantly perturbing the O₂ one, thanks to their concentration differences in biological mediums (∼10⁻¹ μM to ∼10¹ μM for H₂O₂ and ∼10¹ μM to ∼10² μM for O₂) (2, 7–11). Electrochemically active microwire array electrodes as exemplary model systems (Fig. 1C) are experimentally shown to achieve tunable heterogeneities of O₂ and H₂O₂ independently, with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s, and are suitable as a platform for independently perturbing biologically relevant O₂ and H₂O₂ profiles in microbial systems. We further established and experimentally validated two neural networks that inversely design the wire array electrodes’ morphologies toward targeted microenvironments of O₂ and H₂O₂, respectively, which is at least one order of magnitude faster than trial-and-error numerical simulation and two orders of magnitude faster than experimental explorations. The demonstrated inverse design of electrochemically generated controlled gradients not only demonstrates a full electrochemical control of concentration profiles in an electrode’s proximity but also establishes an approach of spatiotemporally mimicking and perturbing extracellular space guided by artificial intelligence.     

F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F₁-ATPase (F₁) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α₃β₃, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F₁ structure and the rotation angle of the motor. Remarkably, a structure of F₁ in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F₁-ATPase.ATP synthase (F₁F_o-ATPase) catalyzes ATP synthesis from ADP and P_i in cells. The isolated F₁ portion is called F₁-ATPase, because it also catalyzes the reverse reaction, ATP hydrolysis (1–3). The α₃β₃γ-catalytic core complex of F₁-ATPase (denoted F₁) is a rotary molecular motor in which three αβ-dimers are arranged around the central γ-shaft (4). Unidirectional rotation of γ is driven by the free energy derived from sequential ATP hydrolysis at catalytic sites in the three αβ-dimers (5–7). Under an external torque, F₁ synthesizes ATP coupled to the rotation of γ in the opposite direction (8). This reversible operation of F₁ is achieved by the tripartite mechanochemical coupling between chemical reactions at the catalytic sites of αβ, conformational changes in the stator ring-α₃β₃, and orientation of γ.A combination of the rotation assay (5, 6) and single-molecule fluorescence imaging techniques (9) has led to a detailed picture of the coupling between chemical reactions in α₃β₃ and the rotary angles (10, 11). One ATP hydrolysis reaction in α₃β₃ drives discrete 80° + 40° substeps of γ in bacterial F₁ (7). The 80° substep is mainly driven by the binding energy of ATP (7, 9). The dwell before the 80° substep is, therefore, named the ATP waiting dwell. Release of the product, ADP, occurs before completion of the 80° substep (9, 10). The angle-dependent affinity of ADP suggests that the ADP release event also contributes part of the energy for the 80° substep (10). The dwell before the 40° substep is called the catalytic dwell; it consists of two rate-limiting events: ATP cleavage and release of the product, P_i (10, 12). The 40° substep is accompanied by a decrease of P_i affinity, with release that, in turn, generates torque (10). The coupling scheme between chemical reactions in α₃β₃ and the rotary angles has, therefore, been almost completely established (10).In the coupling of chemical reactions and α₃β₃-conformations, the key concept is thought to be the binding change mechanism, in which three catalytic sites in F₁ undergo sequential transitions between conformational states with different affinities for nucleotides corresponding to different rotary angles (1). The binding change mechanism is supported by the first crystal structure of F₁, in which two βs adopt the closed form with nucleotides and the other β adopts the open form without a nucleotide (4). The 120° step of γ observed in the rotation assay further supports this mechanism (6).However, we still face significant gaps in the structural ATPase cycle. Previous studies have suggested that F₁ should adopt at least two distinct conformational states for the ATP waiting dwell (ATP waiting form) and the catalytic dwell (catalytic form) based on rotation and tilting angles of γ (13). Furthermore, based on indirect evidence, it has been pointed out that the first crystal structure should represent the catalytic form or forms similar to the catalytic intermediate states (14–18). Although the crystal structures of the α₃β₃γ-complex differ from each other in terms of their nucleotide binding states and detailed configurations of the residues, their global structures are similar to the first crystal structure (19), which leaves the structure of the ATP waiting form unresolved. Closing this gap in the conformational cycle will deepen our understanding of the coupling between chemical reactions, α₃β₃-conformations, conformations, and rotary angles, not least by providing critical input into the theoretical modeling of F₁ (20–26).Here, we use the FRET technique to elucidate the conformational transitions of α₃β₃-conformations in F₁. FRET involves excited-state energy transfer from one fluorescent dye (donor) to another (acceptor) through dipole–dipole interactions (27). Single-pair FRET measurements combined with single-molecule techniques have been used to investigate the dynamics of intramolecular conformational changes or intermolecular interactions at the single-molecule level (28–34), including for F_oF₁-ATP synthase (35–37). We perform single-molecule FRET measurement to monitor distance changes between two fluorescently labeled βs and simultaneously monitor the rotational steps of γ. The FRET data allow us to distinguish the ATP waiting form from the catalytic form and thus, relate these dwelling states to the respective crystal structures. A systematic comparison of the crystal structures reveals the structural basis of the ATPase cycle. This study provides a structural basis for tripartite coupling among chemical reactions, conformations in the stator, and rotary angles in bacterial F₁.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils

Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on ¹³C,¹⁵N-enriched, and fully protonated samples of M₀Aβ_1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and ¹³C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M₀Aβ_1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Amyloid fibrils are highly stable protein deposits found in β-sheet conformations and are notoriously recognized as disruptive agents to cellular function in over 40 human diseases (1, 2). Alzheimer’s disease (AD) is the most pervasive of all known plaque-related diseases and is associated with the presence of amyloid-β (Aβ) peptides in the extracellular space of the brain (3–6). As of 2021, there are ∼6.2 million people in the United States living with Alzheimer’s dementia and ∼50 million worldwide (7), and there is as of yet no cure available for AD. In order to address this epidemic, it is essential that we learn as much as possible about the formation and structure of Aβ plaques, including the detailed features of their catalytic surface, in order to design and develop appropriate treatments to limit the propagation of aggregates and the generation of toxic forms.Aβ is derived from the C-terminal region of the amyloid precursor protein (APP), a membrane protein in neuronal cells, via proteolysis by β- and γ-secretase (8, 9). One of the principal challenges in rationalizing AD etiology is Aβ’s diversity in peptide length, mutations, and posttranslational modifications (10). Their low solubility renders solution NMR ineffective, and high-resolution diffraction analyses have thus far been restricted to shorter peptides with all or most residues being ordered in the fibril core structure (11). Cryogenic electron microscopy (cryo-EM) has made strides in resolution in fibril studies within the past decade (12–18), but faces challenges studying with atomic-level detail due to polymorphism and heterogeneity in the fibril macroassemblies. Studying the individual and collective roles of amyloids at atomic resolution therefore requires alternative, high-resolution, high-throughput techniques for structural analysis. Magic-angle spinning NMR (MAS-NMR) was introduced as a technique with the potential to address these problems (19, 20). Recent technical advances (21, 22) and progress in sample preparation (23) have vastly improved the sensitivity and resolution of the spectra (24). Accordingly, there are now publications describing high-resolution structures of Aβ (25–29) and other amyloid (12, 16, 30–35) fibrils based on distance and torsion angle constraints derived from MAS experiments.To date, all of the known NMR structures of amyloid fibrils were determined using constraints obtained from ¹³C/¹⁵N MAS spectra, which are inhomogeneously broadened and therefore feature well-resolved lines at low spinning frequencies (<25 kHz) (36). However, resolution often remains insufficient for in-depth analysis, and the experiments require relatively large amounts of peptide and extensive signal acquisition periods. Two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP)—promise to circumvent these issues by reducing signal acquisition times or, alternatively, the amount of protein required for the experiment (37). ¹H detection offers a factor of (γ_H/γ_S)^3/2 gain in sensitivity, where S is usually a low γ-spin (38–40) such as ¹³C or ¹⁵N. In these two cases it is possible to achieve a factor of ∼8 or ∼32 gain in sensitivity, respectively. Importantly, ¹H detection also introduces an additional spectral dimension and therefore significantly increases the resolution. In parallel, DNP offers a general approach to enhancing sensitivity by factors of ∼100, dramatically reducing signal acquisition times (by ∼10⁴). It does so by exploiting the high spin polarization of unpaired electrons (of gyromagnetic ratio γ_e ∼660 times larger than γ_H) of a paramagnetic polarizing agent to enhance sensitivity by a theoretical factor of γ_e/γ_H. (41–44) Furthermore, DNP experiments are conducted at ∼100 K, thereby increasing the Boltzmann polarization and sensitivity by another factor of ∼3 over experiments conducted at ambient temperature (45).While these arguments are well established for MAS NMR in many systems, it is less obvious that they are applicable to amyloid samples because spectra of amyloids are known to be broad for a variety of reasons, such as sample purity and polymorphism. Furthermore, ¹H-detected NMR at moderate MAS frequencies (∼20 to 60 kHz) needs to be coupled to different levels of deuteration to ensure high sensitivity and narrow linewidths (42). Accordingly, deuteration with partial reprotonation of the amide or Hα sites has been implemented in pioneering studies on Aβ_1-40 at 20 kHz MAS (46), HET-s(218–289) (47), and D76N-β2m at 60 kHz MAS (48). In addition, selective protonation in Aβ_1-40 fibril methyl groups at 18 kHz MAS has led to highly resolved ¹H-detected ¹³C correlations (49). In deuterated samples, however, the amount of potentially available structural information is significantly reduced, which can impair high-resolution structure determinations. The advent of 0.7 mm MAS rotors that achieve ω_r/2π >110 kHz attenuates ¹H-¹H dipole couplings and allows direct acquisition of multidimensional ¹H data without requiring deuteration (50). Furthermore, the spectra provide assignments and structural information. While a proof-of-concept application of this approach was demonstrated on fully protonated highly regular prion fibrils (51, 52), it is not clear whether this methodology is generally applicable and extendable to the detection of resolved inter- and intramolecular contacts in complex amyloid assemblies.In parallel, our MAS DNP studies on M₀Aβ_1-42 (28, 32, 53) report significant broadening of the NMR lines at cryogenic temperatures, which was attributed to distributions of conformations trapped at low temperature and is therefore inhomogeneous in origin. The loss of resolution associated with the MAS DNP methodology is a major obstacle for the detailed structural study of uniformly labeled amyloid samples. Concurrently, reports of well-resolved spectra at high fields and spinning frequencies suggest that the broadening is homogeneous (54–56). The advent of DNP instrumentation operating at high magnetic fields (18.8 T) and faster MAS (ω_r/2π = 40 kHz) provides an approach to alleviate this limitation by attenuating homogeneous couplings (57). However, this comes at the expense of the enhancement factor, potentially compromising the capacity to carry out expeditious multidimensional and multinuclear correlations. Moreover, NMR spectra of amyloid fibrils are known to suffer from additional debilitating broadening associated with their heterogeneous character (sample purity, polymorphism, etc.), which may mitigate the benefits of high magnetic fields.In this work, we show that high resolution and sensitivity are possible for fibrils of M₀-Aβ_1-42. Notably, we demonstrate rapid resonance assignment and site-resolved detection of numerous site-specific internuclear proximities on submilligram sample quantities via ¹H-detected NMR at ω_r/2π ∼110 kHz and high field (23.4 T/1,000 MHz for ¹H) at room temperature and ¹³C-detected DNP MAS NMR at ω_r/2π = 40 kHz and high field (18.8 T/800 MHz for ¹H) at low temperature. While both ¹H detection and DNP afford increased sensitivity, we estimate, using approaches outlined by Ishii and Tycko (40), that DNP, with our current ε = 22, yields a factor of ∼6.5 higher sensitivity. These results therefore illuminate possible paths for the rapid structure elucidation of amyloid fibrils available in limited quantities.     

Unusual Mott transition in multiferroic PbCrO3

The Mott insulator in correlated electron systems arises from classical Coulomb repulsion between carriers to provide a powerful force for electron localization. Turning such an insulator into a metal, the so-called Mott transition, is commonly achieved by “bandwidth” control or “band filling.” However, both mechanisms deviate from the original concept of Mott, which attributes such a transition to the screening of Coulomb potential and associated lattice contraction. Here, we report a pressure-induced isostructural Mott transition in cubic perovskite PbCrO₃. At the transition pressure of ∼3 GPa, PbCrO₃ exhibits significant collapse in both lattice volume and Coulomb potential. Concurrent with the collapse, it transforms from a hybrid multiferroic insulator to a metal. For the first time to our knowledge, these findings validate the scenario conceived by Mott. Close to the Mott criticality at ∼300 K, fluctuations of the lattice and charge give rise to elastic anomalies and Laudau critical behaviors resembling the classic liquid–gas transition. The anomalously large lattice volume and Coulomb potential in the low-pressure insulating phase are largely associated with the ferroelectric distortion, which is substantially suppressed at high pressures, leading to the first-order phase transition without symmetry breaking.Early transition-metal (TM) oxides with partially filled d electrons are strongly correlated (1, 2). Such correlated systems often present exciting new physics and technologically useful electronic and magnetic properties. Mott transition, characterized by delocalization of d electrons, is an attractive phenomenon for exploring the correlated nature of electrons (2, 3). Since the early failure of band theory in the 1930s, the Coulomb repulsion (U) has been proposed to be a strong force that causes electron localization (4, 5). In such electrostatic interaction, the repulsion energy decreases with the compressed lattice because of the screening effect (5–7). Consequently, as originally predicted by Mott (5), the Mott transition is controlled by U at pressures (P).Despite several decades of intensive study, it is still challenging to experimentally validate this view of Mott transition, because U is experimentally difficult to determine, and for most correlated materials it is independent of the pressure. For the known Mott systems, they are found to be controlled by either the bandwidth [e.g., the organic compound κ-Cl (8–10) and Cr-doped V₂O₃ (11, 12)] or band filling (i.e., doping of charge carriers into the parent insulator) (2). Recently, electronic transitions have frequently been reported in late 3d TM oxides (e.g., MnO) (13–16), which are theoretically attributed to bandwidth control (15) or crystal-field splitting (17). For those oxides, a U-controlled mechanism has also been proposed by Gavriliuk et al. (14) and Gavriliuk and coworkers (18); however, the spin cross-over, instead of the screening effect, is believed to contribute to the decreased U (14, 18). Complicating matter further is that the U of (Mg_1–xFe_x)O was computed to increase with pressures (13).TM oxides with a perovskite structure (ABO₃) often exhibit intriguing structural, magnetic, and electronic properties for the study of correlated systems. Among them, PbCrO₃ is such a material that can only be synthesized at high pressures. At ambient pressure, it adopts a paramagnetic (PM), cubic structure at room temperature (T) with an anomalously large unit-cell volume and transforms to an antiferromagnetic (AFM) ground state at low temperatures (19, 20). The magnetic properties arise from unpaired 3d electrons in Cr (i.e., nominally 3d²) with a large U value of 8.28 eV (19–21). Under high pressures, an isostructural phase transition (i.e., no symmetry breaking) has recently been reported in PbCrO₃ with ∼9.8% volume reduction at ∼1.6 GPa; it is the largest volume reduction known in transition-metal oxides (22). Compared with the low-P phase, the high-P phase possesses a more “normal” unit-cell volume (see refs. 21 and 22) and a moderate U of ∼3 eV (23), suggesting a collapse of Coulomb repulsion energy at the phase transition. Because of the reduced U, the mobility of 3d electrons at high pressures is energetically more favorable, which would lead to d-electron delocalization. Apparently, this is a U-driven Mott transition in PbCrO₃ as conceived by Mott. However, to date, the electronic properties of both PbCrO₃ phases have only poorly been explored. In particular, controversial electronic states, including semiconductor (24, 25), half-metal (21), or insulator (20), have been reported for the low-P phase. Besides, the crystal structure and elastic and magnetic properties, as well as the underlying mechanism for the isostructural transition, are still unsettled issues, calling for rigorous investigation into this material.With these aims, in this work we present a comprehensive study on PbCrO₃ with a focus on the P-induced electronic transition. Our findings unveil a unique Mott transition in this perovskite and a new mechanism underlying the isostructural transition.     

Dual role of CO in the stability of subnano Pt clusters at the Fe3O4(001) surface

Interactions between catalytically active metal particles and reactant gases depend strongly on the particle size, particularly in the subnanometer regime where the addition of just one atom can induce substantial changes in stability, morphology, and reactivity. Here, time-lapse scanning tunneling microscopy (STM) and density functional theory (DFT)-based calculations are used to study how CO exposure affects the stability of Pt adatoms and subnano clusters at the Fe₃O₄(001) surface, a model CO oxidation catalyst. The results reveal that CO plays a dual role: first, it induces mobility among otherwise stable Pt adatoms through the formation of Pt carbonyls (Pt₁–CO), leading to agglomeration into subnano clusters. Second, the presence of the CO stabilizes the smallest clusters against decay at room temperature, significantly modifying the growth kinetics. At elevated temperatures, CO desorption results in a partial redispersion and recovery of the Pt adatom phase.Subnanometer metal particles exhibit a range of interesting electronic or catalytic properties that can vary substantially with the removal or addition of a single atom (1–6). Understanding the mechanistic details underlying the rearrangement of the active phase is important because changes in cluster size and shape are known to be commonplace under the conditions used in heterogeneous catalysis (7, 8), and because such processes are associated with deactivation phenomena such as sintering. Although sintering is usually regarded as a thermally activated process, there is increasing evidence that adsorbates influence sintering rates in a reactive environment by formation of mobile metal-molecule intermediates (2, 8–30). Indeed, in a previous study we demonstrated that the formation of highly mobile Pd₁–CO species led to enhanced sintering in the Pd/Fe₃O₄(001) system (31). Here, we turn our attention to Pt. Mobility is induced in the form of Pt₁–CO. In addition, CO stabilizes the smallest clusters. When it desorbs, Pt dimers break up into single atoms; thus, the CO is necessary for preserving nuclei that act as seeds for further growth. Using room-temperature scanning tunneling microscopy (STM), complemented by X-ray photoelectron spectroscopy (XPS) and density functional theory with an on-site Hubbard U (DFT+U), we follow the CO-induced diffusion and coalescence of Pt atom-by-atom, creating catalytically active (32) subnano clusters with a well-defined size distribution. On heating, desorption of CO leads to significant redispersion of Pt into the adatom phase.     

Massive and rapid predominantly volcanic CO2 emission during the end-Permian mass extinction

The end-Permian mass extinction event (∼252 Mya) is associated with one of the largest global carbon cycle perturbations in the Phanerozoic and is thought to be triggered by the Siberian Traps volcanism. Sizable carbon isotope excursions (CIEs) have been found at numerous sites around the world, suggesting massive quantities of ¹³C-depleted CO₂ input into the ocean and atmosphere system. The exact magnitude and cause of the CIEs, the pace of CO₂ emission, and the total quantity of CO₂, however, remain poorly known. Here, we quantify the CO₂ emission in an Earth system model based on new compound-specific carbon isotope records from the Finnmark Platform and an astronomically tuned age model. By quantitatively comparing the modeled surface ocean pH and boron isotope pH proxy, a massive (∼36,000 Gt C) and rapid emission (∼5 Gt C yr⁻¹) of largely volcanic CO₂ source (∼−15%) is necessary to drive the observed pattern of CIE, the abrupt decline in surface ocean pH, and the extreme global temperature increase. This suggests that the massive amount of greenhouse gases may have pushed the Earth system toward a critical tipping point, beyond which extreme changes in ocean pH and temperature led to irreversible mass extinction. The comparatively amplified CIE observed in higher plant leaf waxes suggests that the surface waters of the Finnmark Platform were likely out of equilibrium with the initial massive centennial-scale release of carbon from the massive Siberian Traps volcanism, supporting the rapidity of carbon injection. Our modeling work reveals that carbon emission pulses are accompanied by organic carbon burial, facilitated by widespread ocean anoxia.

The end-Permian mass extinction (EPME) that occurred at 251.941 ± 0.037 Mya is considered the most severe biodiversity loss in Earth history (1, 2). The EPME coincides with the eruption of the Siberian Traps, a voluminous large igneous province (LIP) that occupies 6 million square kilometers (km²) in Siberia, Russia (3–5). The volcanic activity of this LIP is linked to SO₂ and CO₂ degassing generated by sill intrusion (6–10). The large amount of CO₂ injected into the atmosphere is thought to have led to severe global warming (11–14), catastrophic ocean anoxia (15, 16), and extreme ocean and terrestrial acidification (17–21) being lethal for life on land and in the sea (22). To date, no agreement has been reached regarding the source of the ¹³C-depleted carbon that triggered the global carbon cycle perturbation, the decrease in ocean pH, and the global warming across the EPME. Additionally, atmospheric CO₂ levels following the initial pulse of Siberian Traps volcanism and across the EPME remain poorly known (23, 24), limiting our understanding of the climate feedbacks that occur upon greenhouse gas release during this time.To address this critical gap in our knowledge, we constrain the source, pace and total amount of CO₂ emissions using an Earth system model of intermediate complexity (i.e., carbon centric-Grid Enabled Integrated Earth system model [cGENIE]; SI Appendix) forced by new astronomically tuned δ¹³C records from well-preserved lipid biomarkers preserved in sediments from the Finnmark Platform, Norway. The Finnmark Platform is located offshore northern Norway on the Eastern Barents Sea shelf, hosting an expanded shallow marine section (paleo-water depth roughly 50 to 100 m) where two drill cores were collected (7128/12-U-01 and 7129/10-U-01) spanning the Permian–Triassic transition (Fig. 1). A previously generated bulk organic carbon isotope record (δ¹³C_org) from the same core shows a two-step decline with a total carbon isotope excursion (CIE) magnitude of ∼4‰ (25). Although the sedimentary organic carbon was considered primarily of terrestrial origin, small contributions from marine organic carbon production could not be excluded. Here, we use compound-specific carbon isotope analysis of both long-chain and short-chain n-alkanes preserved in marine sediments in the Finnmark Platform to generate separate yet directly comparable records of δ¹³C for the terrestrial and the marine realm, respectively, across the EPME. Long-chain n-alkanes with a strong odd-over-even predominance (n-C₂₇ and n-C₂₉) are produced by higher plant leaf waxes, and their isotopic composition (δ¹³C_wax) relates to their main carbon source (i.e., atmospheric CO₂) (26). On the other hand, short-chain alkanes (n-C₁₇ and n-C₁₉) are derived from marine algae, and their δ¹³C values (δ¹³C_algae) represent carbon in the marine realm (27, 28). To date, only a few EPME compound-specific carbon isotope studies have been reported, all of which are limited by unfavorable sedimentary facies or high thermal maturity of the organic matter (29, 30). In the present study, the exceptionally low thermal maturity of the organic matter is evident from the yellow color of pollen and spores, indicating a color index 2 out of 7 on the thermal alteration scale of Batten (31), which is equivalent to a vitrinite reflectance R₀ of 0.3%. Moreover, the high sedimentation rate (discussed in Carbon Cycle Quantification Using Astrochronology and Earth System Model) of the siliciclastic sediments at the study site allows for studying both marine and terrestrial CIE across the EPME in unprecedented detail. Taken together, the Finnmark sedimentary records enable the reconstruction of individual yet directly comparable carbon isotope records for the terrestrial and the marine realm that can be astronomically tuned and used to quantitatively assess the source, pace, and total amount of ¹³C-depleted carbon released during the Siberian Traps eruption that led to the EPME. Using our new compound-specific carbon isotope records, rather than marine carbonates, has several advantages: 1) new astrochronology enables a 10⁴-year temporal resolution for our paired marine and terrestrial carbon isotope records; 2) we do not need to assume a constant sedimentation rate between tie point or using diachronous biozones to compare age like those used in global compilations (24) (see Fig. 4A); 3) the δ¹³C_algae data are not artificially smoothed as in ref. 32 to avoid underestimation of the CIE magnitude; and 4) our records are not affected by dissolution or truncation, a phenomenon common to shallow marine carbonates due to the presumed ocean acidification occurred during the EPME (18, 33). In addition, the directly comparable records of δ¹³C for the atmosphere and the ocean offer further insights into the size of the true CIE and rate and duration of carbon emissions.Open in a separate windowFig. 1.(A) Paleogeographical map of the Late Permian, with former and current coastlines. Indicated are 1) the location of Finnmark cores 7128/12-U-01 and 7129/10-U-01, 2) the East Greenland site at Kap Stosch discussed in ref. 52, 3) the GSSP site for the base of the Triassic at Meishan, China, and 4) the Kuh-e-Ali Bashi site of Iran (66, 107). The map was modified after ref. 61. (B) Paleogeography and paleobathymetry of the Late Permian used in cGENIE.Open in a separate windowFig. 4.Synthesized proxy records of carbon isotopes from marine carbonates and fossil C₃ land plants remains, sea surface temperature, and pH. (A) Comparison between δ¹³C_algae and global marine carbonate carbon isotopes from sites at Abadeh, Kuh-e-Ali Bashi, Shahreza, and Zal in Iran, Meishan, Wenbudangsang, and Yanggou in South China, at Bálvány North in Hungary, and at Nhi Tao in Vietnam (24). (B) Comparison between δ¹³C_leaf wax and the δ¹³C of sedimentary leaf cuticles and wood of C₃ land plants from South China (24). (C) Reconstructed sea surface temperature data using conodont fossils (circles) (24) and brachiopods (triangles) (14). The conodont-based temperature data are from sites in the Paleo-Tethys, including Chanakhchi, Kuh-e Ali Bashi, Meishan, Shangsi, and Zal. (D) Relative changes in sea surface pH based on boron isotope proxy from ref. 17 and ref. 20. Pink and red circles are data from scenario 1 and scenario 2 in ref. 17, and green and blue diamonds are data from scenario 1 and scenario 2 in ref. 20.     

Platelet Gi protein Gαi2 is an essential mediator of thrombo-inflammatory organ damage in mice

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The G_i protein Gα_i2 mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα_i2 exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα_i2 in megakaryocytes and platelets (Gnai2^fl/fl/PF4-Cre mice) and found bleeding defects comparable to those in global Gα_i2-deficient mice. To examine the impact of platelet Gα_i2 in postischemic thrombo-inflammatory infarct progression, Gnai2^fl/fl/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gnai2^fl/fl/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gnai2^fl/fl/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα_i2 not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.Platelet activation at sites of vascular injury is essential for normal hemostasis but also is a major pathomechanism underlying acute ischemic disease states such as stroke or myocardial infarction, which represent leading causes of death and severe disability worldwide (1–3).Upon vascular injury, exposed extracellular matrix constituents of the damaged vessel wall allow initial adhesion of platelets, initiating intracellular signaling cascades that result in platelets’ firm adhesion and aggregation (2, 3). Activated platelets deliver diffusible local mediators, such as ADP or thromboxane A₂, to recruit and activate additional platelets into the growing thrombus. Hence, ADP potentiates the aggregatory effects of other stimuli such as thrombin and collagen and thereby contributes to stable thrombus formation. These mediators orchestrate platelet signaling by activating G protein-coupled receptors (GPCRs) (4). In particular, platelet activation by ADP is mediated by two GPCRs, P2Y₁, which couples to the heterotrimeric G protein G_q, and P2Y₁₂, which couples to G_i proteins (5). Deficiency of either P2Y₁ or P2Y₁₂ receptors leads to a reduced aggregation response following ADP stimulation, suggesting a complementary function of the two types of G proteins, G_q and G_i, in the induction of platelet activation (6–9).Only P2Y₁₂ receptors are therapeutically targeted by antagonists, which inhibit platelet aggregation in patients (10, 11). Correspondingly, mice lacking the P2Y₁₂ receptor exhibit a profound defect in platelet activation (7, 8) with prolonged bleeding times which correlate with impaired formation and stability of thrombi. However, the impact of this pathway on the progression of thrombo-inflammatory infarcts in the postischemic brain and heart is unknown. The P2Y₁₂ receptor is reported to signal selectively through the G protein G_i2, although biochemical reconstitution experiments suggest that it interacts with other G_i isoforms such as G_i3 (11–13). Platelet G_i2, however, may interact not only with P2Y₁₂ but also with additional GPCRs present in platelets. Regardless of these considerations, and unlike murine platelets deficient in P2Y₁₂ receptors, Gα_i2-deficient platelets show only a moderate inhibition of platelet aggregation in vitro, and the translation of this defect into the in vivo situation has not been reported thus far (14, 15).A different approach to study how Gα_i2 affects in vivo thrombotic activity of platelets came from a knockin mouse line in which regulator of G-protein signaling (RGS)-insensitive Gα_i2 (G184S) was expressed (16). However, the complexity and severity of the phenotype is likely to limit further studies on the progression of platelet-dependent thrombo-inflammatory infarcts (16).Here, we show that Gα_i3 only partially compensates for the loss of Gα_i2, revealing that platelet Gα_i2 plays a dual role in both thrombus formation in injured vessels and in progression of tissue damage after focal cerebral ischemia or myocardial infarction.     

Molecular basis for lipid recognition by the prostaglandin D2 receptor CRTH2

Prostaglandin D₂ (PGD₂) signals through the G protein–coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD₂ derivative, 15R-methyl-PGD₂ (15mPGD₂), by serial femtosecond crystallography. The structure revealed a “polar group in”–binding mode of 15mPGD₂ contrasting the “polar group out”–binding mode of PGE₂ in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.

Eicosanoids constitute a group of signaling lipid mediators that are derived from arachidonic acid or other polyunsaturated fatty acids (1, 2). As the name indicates, they all possess a long hydrocarbon chain with 20 carbon units, which is usually attached to a carboxylic acid head group. Prostanoids including prostaglandins D₂, E₂, and F₂ (PGD₂, PGE₂, and PGF₂); prostacyclin (PGI₂); thromboxane A₂ (TXA₂) and leukotrienes including leukotriene B₄, C₄, and D₄ (LTB₄, LTC₄, and LTD₄) are representative endogenous eicosanoids that can induce signaling through G protein–coupled receptors (GPCRs) to play critical roles in inflammation, immunity, hemostasis, and tissue repair (1, 3–5). In humans, nine prostanoid receptors have been identified: PGD₂ receptors 1 and 2 (DP1 and DP2), PGE₂ receptors 1 to 4 (EP1 to EP4), PGF₂ receptor (FP), PGI₂ receptor (IP), and TXA₂ receptor (TP) (5). All of these receptors, except for DP2, belong to the α-branch of Class A GPCRs as close phylogenetic neighbors of aminergic receptors (5, 6). DP2, chemoattractant receptor–homologous molecule expressed on type 2 helper T cells (Th2) (also named CRTH2), is more closely related to a group of chemotactic GPCRs in the γ-branch of Class A GPCRs, including chemokine receptors and receptors for LTB₄, formylpeptides, and complement peptides C3a and C5a (6, 7).Certain types of immune cells, including eosinophils, innate lymphoid cells, and Th2 cells, express high levels of CRTH2 (8–11). PGD₂ signaling through CRTH2 can induce chemotaxis of these immune cells, which is a major pathway that drives the onset of type 2 inflammation (11–15). Therefore, the roles of PGD₂–CRTH2 signaling axis in type 2 inflammation-related diseases such as asthma and allergic rhinitis have attracted outstanding research interest (14, 16, 17). CRTH2 antagonists hold the potential of being a new class of anti-inflammatory drugs (16, 18–24). Although clinical trials have generated mixed results on different CRTH2 antagonists for asthma (17–20, 25), it is likely that a certain subpopulation of patients, such as those with a high baseline of Th2 cells or eosinophils, may benefit most from anti-CRTH2 therapy (17, 24). In addition, it has been proposed that insurmountable CRTH2 antagonists with prolonged receptor residence time can provide better, therapeutic efficacy, compared to reversible and fast, dissociating CRTH2 antagonists, which needs further investigation (26).We have previously reported crystal structures of CRTH2 bound to two antagonists, CAY10471 and fevipiprant, which revealed a positively charged environment of the ligand-binding pocket and a potential ligand entry port (27). The structural analysis of antagonist-bound CRTH2 suggested that PGD₂ may enter the ligand-binding pocket through the ligand entry port by opposite charge attraction (27). Recently, crystal structures of several other prostanoid receptors, EP3, EP4, and TP, and cryogenic electron microscopy (cryo-EM) structures of the EP2 and EP4 signaling complexes with PGE₂ have also been reported (28–33). To further study the binding of lipid agonists to CRTH2 and investigate if CRTH2 differs from other prostanoid receptors in lipid recognition and receptor activation, we determined a 2.6-Å resolution, room temperature crystal structure of CRTH2 bound to a PGD₂ derivative by serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) source. We performed computational simulation studies using the CRTH2–15R-methyl-PGD₂ (15mPGD₂) structure, which helped identify the roles of specific residues surrounding the ligand entry port and provided molecular insights into the events that could facilitate ligand binding. Results from our mutational analysis of the identified residues helped us further strengthen the proposed model for ligand capture and entry in CRTH2.     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection

A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate V_H3 plasmablast expansions and increased V_H3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate V_H3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4⁺ T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and V_H3 variant heavy chains of Ig. V_H3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. V_H3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of V_H3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses.The gram-positive pathogen Staphylococcus aureus evolved to colonize humans and their domesticated animals (1). The nares, skin, and gastrointestinal tract of approximately one-third of the human population are stably colonized, whereas another third is intermittently colonized (2). Colonization increases the risk of S. aureus infection, specifically for skin and soft tissue infection, invasive disease, as well as bacteremia (3, 4). S. aureus infection leads to the formation of abscess lesions supporting pathogen replication and dissemination (5). Even with antibiotic and surgical therapy (drainage of abscess lesions), S. aureus persists and causes recurrent disease, manifesting as relapse infections by strains responsible for the index case (6, 7). S. aureus persistence, recurrent disease, and the overall outcome of infections (morbidity and mortality) are impacted by the development of antibiotic-resistant strains, designated MRSA (methicillin-resistant S. aureus) (8). The emergence of MRSA strains has led to dramatic increases in the mortality of S. aureus infections, highlighting the need for development of new therapeutic and preventive strategies (9, 10).S. aureus-infected individuals display expansions of blood V_H3 clonal plasmablasts and V_H3 clonal antibodies (11). Staphylococcal protein A (SpA), a cell wall-anchored molecule that is also released from the bacterial surface, associates with the variant heavy chains of V_H3 clonal IgG and IgM (B-cell receptors) (12, 13). Earlier work reported that SpA on bacterial surfaces but also SpA purified from S. aureus cell walls stimulate the proliferation of human peripheral blood B lymphocytes (14). In contrast, injection of recombinant SpA, purified from Escherichia coli, into mice induced trafficking and apoptosis of marginal zone B cells and innate-like B1 cells (15–17). Persistent depletion of functional B-cell repertoires suggested that animals treated with recombinant SpA may be more susceptible to infections with blood-borne pathogens (18). In agreement with these observations, injection of recombinant SpA into mice with systemic lupus erythematosus (SLE) depletes B1 and marginal zone B cells to alleviate antibody-induced nephritis and renal failure associated with SLE (19).We sought to address the apparent paradox between V_H3 clonal B-cell expansion during S. aureus infection in humans and the collapse of V_H3 clonal B cells in mice treated with recombinant SpA. Earlier work demonstrated that S. aureus sortase A cleaves SpA precursors and covalently links the C-terminal end of polypeptides to peptidoglycan cross-bridges in the cell-wall envelope (20, 21). LytM hydrolase cuts the staphylococcal cell wall to release peptidoglycan-linked SpA into the extracellular medium (22). We show here that peptidoglycan-linked SpA, but not recombinant SpA, induces V_H3 clonal antibody expansions in mice.