A family of starch-active polysaccharide monooxygenases

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1–5). They have recently been found to oxidatively degrade chitin (6–8) and cellulose (8–14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10–12, 14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9–12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved monocopper active site on a flat protein surface (Fig. 1A) (2, 6, 7, 9, 10, 15–17). Two histidine residues in a motif termed the histidine brace coordinate the copper center. The N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the N_ε position in fungal PMOs (Fig. 1A).Open in a separate windowFig. 1.(A) Representative overall and active site structures of fungal PMOs (PDB ID code 2YET) (10). (B) Structure of cellulose (18, 19). Chitin also contains β(1→4) linkages and has similar crystalline higher order structure to cellulose. (C) Model structure of amylopectin (23–25). Hydrogen bonds are shown with green dashed lines.Considering the conserved structural features, it is not surprising that the currently known PMOs act on substrates with similar structures. Cellulose and chitin contain long linear chains of β(1→4) linked glucose units and N-acetylglucosamine units, respectively (Fig. 1B). The polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures (18–21). PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6, 16, 22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8, 13, 14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.Here, we report on the identification of new families of PMOs that contain several key features of previously characterized PMOs, but act on substrates different from cellulose or chitin. A member of one of these novel families of PMOs, NCU08746, was shown to oxidatively cleave amylose, amylopectin, and starch. We designate the NCU08746 family as starch-active PMOs. Both amylose and amylopectin contain linear chains of α(1→4) linked glucose, whereas the latter also contains α(1→6) glycosidic linkages at branch points in the otherwise α(1→4) linked polymer. Unlike cellulose and chitin, amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms (23–27) (see Fig. 1C for example). Starch exists partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose. The discovery of starch-active PMOs shows that this oxidative mechanism of glycosidic bond cleavage is more widespread than initially expected.     

Tuning a riboswitch response through structural extension of a pseudoknot

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ₁-I) and II (preQ₁-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ₁-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ₁-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ₁-II riboswitches remain unclear.PreQ₁ riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ₁), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ₁-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ₁ metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ₁-II riboswitch represents a putative target for antibiotic intervention. Although preQ₁ class I (preQ₁-I) riboswitches have been extensively investigated (18–28), preQ₁ class II (preQ₁-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ₁-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ₁ with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ₁ class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ₁); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ₁-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ₁-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ₁-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.     

Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.Leukocyte extravasation from blood to sites of infection and inflammation or to specific organs is achieved by a sequential adhesion cascade: (i) rolling, (ii) chemokine-induced activation, (iii) firm adhesion, and (iv) transcellular migration. Rolling is mediated by specialized cell surface adhesion molecules, such as selectins, CD44, and specific types of integrins (1, 2).Under conditions of hydrodynamic flow, receptor–ligand bonds are subjected to tensile mechanical force, which disrupts the receptor–ligand bond (Fig. 1A). In general, the lifetime of the receptor–ligand bond exponentially decreases with an increase of the mechanical force (3). However, there is growing evidence demonstrating that the lifetimes of some receptor–ligand bonds increase when moderate levels of force are applied (4–9). However, the underlying mechanism of this phenomenon is still elusive and in some cases controversial. For example, integrin and bacterial adhesin FimH-mediated adhesion have been explained by an “allosteric model,” in which mechanical force induces allosteric changes of the receptor, resulting in the stabilization of the high-affinity state (10, 11). Although selectin-mediated adhesion has been explained by the allosteric model (12), a different “sliding-rebinding model” was also reported (13). This model proposes that force tilts the binding interface to make it parallel to the direction of force, allowing the selectin ligand to slide on the selectin and to form new contacts. The sliding-rebinding model has also been used to explain the force-induced activation of von Willebrand factor-mediated adhesion and actin depolymerization (6, 8).Fig. 1.The effect of the tensile force on the two-state conformations of CD44 HABD. (A) Illustration of the tensile force applied between the receptor on the cells and the immobilized ligand under the fluid shear force. (B) The crystal structure of CD44 HABD ...CD44 is a transmembrane receptor for hyaluronan (HA) (14). CD44–HA interactions are involved in various physiological and pathological processes mediated over a wide range of hydrodynamic forces, including T-lymphocyte trafficking on the endothelium (15, 16), hematopoietic progenitor cell homing into bone marrow niches (17), and the progression of atherosclerosis (18). The HA-binding domain (HABD) of CD44 adopts two distinctive conformations representing the low- and high-affinity states for HA (19–21). HABD is composed of a conserved Link module and the N- and C-terminal extension segment (22). In the ordered (O) state, the C-terminal segment is well folded (Fig. 1B) (19), whereas it becomes disordered in the partially disordered (PD) state upon ligand binding (Fig. 1C) (20). In addition, solution NMR analyses demonstrated that HABD exists in an equilibrium between the O and PD states in both the HA-unbound and HA-bound states, with a transition rate of ∼500 ms, and that HA binding induces an equilibrium shift toward the PD state (21) (Fig. 1D). The Y161A mutant, which constitutively adopts the PD state, exhibits a higher affinity than wild-type HABD, indicating that the O and PD states represent the low- and high-affinity states for HA, respectively (21) (Fig. 1E). Cells expressing the Y161A mutant exhibited firm adhesion and impaired rolling on an HA substrate, suggesting that the two-state conformations are essential for the CD44-mediated rolling under flow conditions (21).Despite the importance of the mechanical force in rolling, the means by which it affects the CD44-mediated rolling remain poorly characterized. Recently, it was reported that the rolling of CD44-expressing cells is enhanced at the higher shear stress (23), raising the possibility that CD44 possesses some mechanochemical specializations to resist higher tensile force. Considering the fact that the C terminus of CD44 HABD is connected to the plasma membrane, the force applied from the C terminus of HABD would induce the allosteric transition from the O to the PD state, thereby providing the resistance to the applied force. On the other hand, our previous NMR studies demonstrated that more than 90% of HABD adopts the PD state in the HA-bound state (21), indicating that the free energy of the PD state can be lowered upon HA binding, regardless of the presence or absence of the tensile force. Therefore, it is worthwhile to investigate whether the CD44–HA interaction is strengthened by the tensile force.To assess the effect of the tensile force on the CD44-mediated rolling, we established a cell-free rolling system using cell-sized beads, which are coated with recombinant HABDs. The effect of the tensile force can be investigated by comparing the rolling activity of the beads coated with the ligand-binding domain via the N-terminal or the C-terminal tag (Fig. 1G) (10). We compared the rolling behavior of the beads with N- or C-terminally attached HABD and found that the rolling behavior was stabilized only at higher shear stress, when HABD was attached to the beads via the C-terminal tag. Steered molecular dynamics (SMD) simulations suggested that the force from the C terminus induces the dissociation of the “mechanosensitive latch” in the C-terminal region, which triggers the conversion from the O to the PD state. Based on these results, we propose that the tensile force from the C terminus stabilizes the CD44–HA bond by inducing a rapid transition from the O to the PD state, thereby sustaining the CD44-mediated cell rolling under higher shear stress conditions.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.Muscle contraction is driven by the relative sliding of the actin-containing thin filaments along the myosin-containing thick filaments arranged in a parallel array in the muscle sarcomere (Fig. 1A). Filament sliding in turn is driven by a structural change in the myosin head domains (Fig. 1B) while they are bound to actin, coupled to the hydrolysis of ATP (1). Contraction of skeletal and cardiac muscle is triggered by calcium binding to troponin in the thin filaments, accompanied by a change in the structure of the thin filaments that permits myosin head binding (2). However, the strength and dynamics of contraction are modulated by posttranslational modifications in other sarcomeric proteins, including the myosin regulatory light chain (RLC) (3), which is part of the myosin head, and myosin binding protein-C (4–6) (MyBP-C) (Fig. 1B). In an emerging concept of thick filament regulation in striated muscle that is analogous to myosin-linked regulation in smooth muscle (7–11), RLC and MyBP-C are thought to modulate contraction by controlling the conformation of the myosin heads.Open in a separate windowFig. 1.Sarcomere location and domain architecture of MyBP-C. (A) C-zone (green) of the thick filament in relation to its proximal (P) and distal (D) regions and the thin filament (gray). (B) Cartoon representation of MyBP-C (green) anchored to the thick filament backbone (purple) via its C-terminal domains; myosin heads are pink and troponin is yellow. (C) Domain organization and interactions of MyBP-C.According to this concept, the thick filament has an OFF state in which the myosin heads are folded back against its surface (Fig. 1B), rendering them unavailable for interaction with actin, and an ON state in which the heads are released from the thick filament surface and made available for actin binding. The physiological and pathological significance of thick filament regulation and its relationship to the well-studied thin filament mechanisms remain poorly understood, but much recent attention has focused on MyBP-C for two main reasons. First, mutations in the cardiac MYBPC3 gene are commonly associated with hypertrophic cardiomyopathy (12, 13), and this association has driven a wide range of studies at the molecular, cellular, and whole-animal levels aimed at understanding the etiology of MYBPC3-linked disease. Second, although MyBP-C is a constitutive component of the thick filament, there is a large body of evidence that it can also bind the thin filaments (14, 15), raising the possibility that one role of MyBP-C may be to synchronize the regulatory states of the thin and thick filaments (11, 15–17).MyBP-C is localized to the central region or “C-zone” of each half-thick filament (Fig. 1A), appearing in nine transverse stripes with a 43-nm periodicity closely matching that of the myosin heads (Fig. 1B) (10). MyBP-C has 11 Ig-like or fibronectin-like domains (Fig. 1C) denoted C0–C10, with additional linking sequences, notably the MyBP-C “motif” or “m” domain between C1 and C2 and the proline/alanine-rich (P/A) linker between C0 and C1. The m domain has multiple phosphorylation sites (4–6). Constitutive binding to the thick filament is mediated by interactions of domains C8–C10 with myosin and titin. The C1mC2 region binds to the coiled-coil subfragment-2 (S2) domain of myosin adjacent to the myosin heads, and this interaction is abolished by MyBP-C phosphorylation (5); the C0 domain binds to the RLC in the myosin head itself (18). The N-terminal domains of MyBP-C also bind to actin in a phosphorylation-dependent manner (14, 15) (Fig. 1B), and EM and X-ray studies on intact sarcomeres of skeletal muscle suggest that MyBP-C binds to thin filaments under relaxing conditions (10, 11).The function of MyBP-C and the mechanisms underlying its modulation in cardiomyopathy remain poorly understood, however. Ablation of MyBP-C in a knockout mouse model leads to a hypertrophic phenotype associated with impaired contractile function (19), but cardiomyocytes isolated from these mice exhibit increased power output during working contractions (20). A range of studies at the isolated protein and cellular levels have led to the concept that MyBP-C exerts a predominantly inhibitory effect on contractility mediated through two distinct mechanisms (15, 16, 21). MyBP-C may tether myosin heads to the surface of the thick filament, preventing their interaction with actin, and its N terminus may bind to thin filaments, inhibiting interfilament sliding at low load. Other studies, however, have demonstrated an activating effect of MyBP-C mediated by binding of its N-terminal domains to the thin filament. N-terminal fragments of MyBP-C enhance force production in skinned cardiac muscle cells and motility in isolated filament preparations at zero or submaximal calcium concentrations (22–25). The same effect is observed in cardiomyocytes from MyBP-C knockout mice (22), suggesting that the activating effect is not due to competitive removal of an inhibitory effect of native MyBP-C.To resolve these apparently contradictory hypotheses about the physiological function of the N-terminal domains of MyBP-C, we determined the structural changes in the thick and thin filaments of intact sarcomeres in heart muscle cells induced by N-terminal MyBP-C fragments using bifunctional rhodamine probes on RLC and troponin C (TnC) (26). These probes allowed the structural changes in both types of filament to be directly compared with those associated with calcium activation and myosin head binding in the native environment of the cardiac muscle sarcomere. The results lead to a model for the physiological function of MyBP-C that integrates the regulatory roles of the thin and thick filaments and the inhibitory and activating effects of MyBP-C at the level of the intact sarcomere.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Robust excitons inhabit soft supramolecular nanotubes

Nature''s highly efficient light-harvesting antennae, such as those found in green sulfur bacteria, consist of supramolecular building blocks that self-assemble into a hierarchy of close-packed structures. In an effort to mimic the fundamental processes that govern nature’s efficient systems, it is important to elucidate the role of each level of hierarchy: from molecule, to supramolecular building block, to close-packed building blocks. Here, we study the impact of hierarchical structure. We present a model system that mirrors nature’s complexity: cylinders self-assembled from cyanine-dye molecules. Our work reveals that even though close-packing may alter the cylinders’ soft mesoscopic structure, robust delocalized excitons are retained: Internal order and strong excitation-transfer interactions—prerequisites for efficient energy transport—are both maintained. Our results suggest that the cylindrical geometry strongly favors robust excitons; it presents a rational design that is potentially key to nature’s high efficiency, allowing construction of efficient light-harvesting devices even from soft, supramolecular materials.The most remarkable materials that demonstrate the ability to capture solar energy are natural photosynthetic systems such as those found in primitive marine algae and bacteria (1–10). Their light-harvesting (LH) antennae are crucial components, because they absorb the light and direct the resulting excitation energy efficiently to a reaction center, which then converts these excitations (excitons) into charge-separated states (1, 4, 11, 12). Although the noncovalent interactions that link the individual molecules within the LH antennae are weak, the excitation transfer interactions between the molecules are relatively strong; new excited states, so-called Frenkel excitons (13), are generated that are delocalized over a number of molecules (1). These delocalized excitons are key to nature’s efficiency and are therefore of high interest (14–20).To create such efficient LH systems, nature assembles molecular subunits into individual supramolecular structures, which are then further assembled into close-packed superstructures (1, 4, 7–10, 12, 21). This hierarchical assembly is a generic motif of nature’s photosynthetic systems. As with natural systems, assembling artificial LH devices from supramolecular structures will require close packing into hierarchical assemblies to maximize the amount of absorbed light (19). Therefore, key to our ability to tune materials properties for efficient LH applications is a basic understanding of the role of each level of the hierarchy: from the individual molecule, to the individual supramolecular building block, to the close-packed assembly. Whereas the role of the individual molecules in the excitonic properties of the building blocks is well-studied (1, 7–10, 20, 22–37), the effect of structural hierarchy remains an open question because the system’s soft structure may be easily altered upon close packing: Do the excitonic properties change as the individual supramolecular structures assemble into a close-packed superstructure? In other words, can we simplify the materials properties as a superposition of the properties of the building blocks or does their close packing require a different interpretation?In nano-science size and shape have important impact on a system’s properties (1). Interestingly, one of nature’s most spectacular class of LH antennae systems that was found in green sulfur bacteria (7–10, 15, 34, 38, 39) uses cylindrical-shaped geometries as a basic design principle for their supramolecular building blocks (7–10). Does nature gain an advantage from using cylindrical-shaped rather than simple sheet-like geometries as a design principle for one of its most efficient LH antennae?The ultimate next step is to elucidate the impact of hierarchical assembly on excitonic properties that are critical to the system’s efficiency. However, it is difficult to disassemble nature’s hierarchical structures into their supramolecular building blocks to study and understand the impact of hierarchical assembly. A promising approach would be to use a well-defined model system for which basic investigations are possible in every single step on the hierarchical assembling process.In this paper we realize the comprehensive investigation of an artificial model system that mirrors the complexity of natural LH antennae: artificial LH nanotubes (LHNTs) of amphiphilic cyanine dye molecules (40) self-assembled into a cylindrical geometry with a diameter on the order of 10 nm (41) and a length that may extend to micrometers (29, 42). We show that these LHNTs can complete the hierarchical assembly by further assembling into bundled LHNTs that consist of close-packed cylindrical building blocks. We use a cross-disciplinary approach that combines rigorous structural characterization along with optical spectroscopy through complementary experimental methods—electron microscopy techniques, linear and nonlinear optical spectroscopy, flash dilution, and redox-chemistry—and theoretical simulations. This approach allows for a direct comparison of the excitonic properties originating from the individual supramolecular building blocks and from the close-packed building blocks. The observed spectral changes upon close packing can be explained by minor changes within the supramolecular structure of the building blocks. In contrast, we find that the properties prerequisite for efficient excitation energy transport are maintained through close packing: a high degree of internal order, strong excitation transfer interactions, and large exciton delocalization, evoking the term “robust excitons.” Our results suggest that the cylindrical geometry presents a rational design that is potentially key for constructing efficient LH systems from artificial supramolecular materials, potentially by minimizing perturbations of the excitonic properties upon formation of hierarchical structures.In particular, we study LHNTs (31) composed of the amphiphilic cyanine dye C8S3 (Fig. 1A, Inset and Materials and Methods). Cryo-electron microscopy (cryo-EM) shows that freshly prepared LHNTs in water/methanol solution are well separated from one another (Fig. 1 A and B), consistent with previous work (31, 41, 43). These LHNTs present a homogeneous ensemble with a remarkably uniform supramolecular structure (29) and are composed of two concentric cylinders, as schematically illustrated in Fig. 1C, separated by about 4 nm: an inner cylinder with a diameter of ∼6 nm (43) and an outer cylinder with a diameter of ∼13 nm (41, 43). Cryo-EM reveals that the double-walled LHNTs further self-assemble over time into bundled nanotubular structures (Fig. 1D). Bundles are either straight or twisted (Fig. 1E and SI Appendix, section 1).Open in a separate windowFig. 1.Correlating morphological and optical properties. (A) Cryo-EM micrograph of double-walled LHNTs self-assembled from amphiphilic cyanine dye molecules (abbreviated as C8S3; Materials and Methods) with molecular structure in inset. (B) EM micrograph at higher magnification shows the double-walled nature of the LHNTs. (C) Schematic illustrates the LHNT consisting of two concentric cylinders self-assembled from C8S3 molecules (for clarity using only one molecule per unit cell) with the hydrophilic sulfonate groups (red) on the exterior, the hydrophobic alkyl chains (light gray) in the interior of the bilayer, and the cyanine dye chromophore (dark gray) in the middle. (D) Cryo-EM micrograph of bundled LHNTs in overview and (E) at higher magnification shows three examples of different morphologies: bundles with a straight morphology (right) and with increasing amounts of twist (middle and left), which is indicated by the Moiré pattern (i.e., alternating bands of sharp and blurred lines along the cylindrical axis). (F) Absorption spectra. Black: absorption spectrum of C8S3 dye molecules (monomers) dissolved in methanol. Red: absorption spectrum upon self-assembling of double-walled LHNTs prepared in water/methanol. Blue: absorption spectrum of bundles of LHNTs. || and ⊥ indicate polarization parallel and perpendicular to the cylinder axis, respectively. See text for transition labeling and SI Appendix, section 3 for polarization of the bands.Upon self-assembly into double-walled LHNTs the broad absorption band of the monomers (Fig. 1F) undergoes a large redshift of ∼80 nm (∼2,500 cm⁻¹), reflecting strong excitation transfer interactions between the molecular transition dipole moments of the molecular subunits (31). The narrowing of the absorption peaks relative to the monomer spectrum, so-called exchange narrowing (13), originates from the delocalization of the excitation over multiple molecular subunits. These delocalized excitations average out short-range monomer inhomogeneities. The exciton delocalization length is defined as the number of molecular subunits over which the excitation is delocalized. In addition, the cylindrical geometry gives rise to a complex pattern of absorption bands (25) that has previously been mapped onto the system’s structure (Fig. 1F): Bands 1 and 3 mainly originate from the inner cylinder, whereas band 2 mainly originates from the outer cylinder (31). The narrow linewidth of low-energy band 1 suggests large exciton delocalization (23, 44).Interestingly, monitoring the bundling process in real time by means of absorption spectroscopy (SI Appendix, section 2) shows that the double-walled LHNTs and the bundled LHNTs have different optical properties. Upon bundling the two main absorption bands of the double-walled LHNTs at 599 nm (band 1) and at 589 nm (band 2) disappear (Fig. 1F), and two new transitions grow in: a narrow band at 603 nm (I) and a broad band at 575 nm (II). We assign these two new bands to the bundled LHNTs. For the double-walled LHNTs (Fig. 1F), bands 1 and 2 are polarized primarily parallel to the cylindrical axis, whereas band 3 is polarized primarily perpendicular to it. For the bundled LHNTs (Fig. 1F), band I is primarily polarized parallel whereas the broad band II is primarily polarized perpendicular (SI Appendix, section 3).     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1

The cariogenic bacterium Streptococcus mutans uses adhesin P1 to adhere to tooth surfaces, extracellular matrix components, and other bacteria. A composite model of P1 based on partial crystal structures revealed an unusual complex architecture in which the protein forms an elongated hybrid alpha/polyproline type II helical stalk by folding back on itself to display a globular head at the apex and a globular C-terminal region at the base. The structure of P1’s N terminus and the nature of its critical interaction with the C-terminal region remained unknown, however. We have cocrystallized a stable complex of recombinant N- and C-terminal fragments and here describe a previously unidentified topological fold in which these widely discontinuous domains are intimately associated. The structure reveals that the N terminus forms a stabilizing scaffold by wrapping behind the base of P1’s elongated stalk and physically “locking” it into place. The structure is stabilized through a highly favorable ΔG_solvation on complex formation, along with extensive hydrogen bonding. We confirm the functional relevance of this intramolecular interaction using differential scanning calorimetry and circular dichroism to show that disruption of the proper spacing of residues 989–1001 impedes folding and diminishes stability of the full-length molecule, including the stalk. Our findings clarify previously unexplained functional and antigenic properties of P1.Streptococcus mutans is a recognized cause of human dental caries (cavities), the most common infectious disease worldwide (1). Identifying how S. mutans interacts with host components at the molecular level is essential to fully understand its virulence properties. The sucrose-independent adhesin P1 (AgI/II, antigen B, PAc) is localized on the surface of this oral pathogen, along with many other streptococci (2–7). In the oral cavity, S. mutans P1 interacts with the salivary agglutinin glycoprotein complex composed predominantly of scavenger receptor gp340/DMBT1 (2, 3, 5–10). Without a complete structural model, the mechanisms by which P1 binds to host components have not yet been fully characterized.P1’s primary structure (Fig. 1A) contains a 38-residue signal sequence, the heretofore uncharacterized N-terminal region, three alanine-rich repeats (A1–3), a central domain containing a so-called variable (V) region (11), three proline-rich repeats (P1–3), a C-terminal region consisting of three domains (C1–3), an LPxTG sortase-recognition motif, and wall- and membrane-spanning regions (12, 13). Recent partial X-ray crystal structure and velocity centrifugation studies of the intact protein unveiled a unique architecture in which the ∼185-kDa (1,561-aa) protein folds back on itself to form a ∼50-nm elongated hybrid helical stalk that separates two independent adherence domains, with a globular head at the apex and a globular C-terminal region at the base (13–15) (Fig. 1B).Open in a separate windowFig. 1.Schematic representation of the primary and modeled tertiary structure of P1. (A) Primary structure of P1 and location of polypeptides used in this study. (B) Proposed tertiary model of P1 based on velocity centrifugation and crystal structures of A3VP1 and C123 fragments (13). (C) Diagram showing the locations of the two engineered Cla1 sites (circled in red) that added isoleucine and aspartic acid residues to either side of the proline-rich region (20).The crystal structure of the third alanine-rich repeat through the first proline-rich repeat first revealed the unusual interaction between the A and P regions to form a hybrid alpha/polyproline type II helix (14). In this model, a globular β super sandwich domain sits at the apex of the molecule (15). At the other end of the hybrid helix, the three contiguous domains of the C-terminal region each adopt a DE-variant Ig-like (DEv-IgG) fold stabilized by isopeptide bonds (13, 16). Despite this recent progress, the structure of the ∼20-kDa N terminus of P1 remained unknown, however (Fig. 1B). We previously demonstrated that proper folding and function of P1 on the surface of S. mutans requires an interaction between N- and C-terminal segments (17), thus increasing the imperative to elucidate the structure of the N terminus in complex with its intramolecular binding partner.For this, we used the recombinant N-terminal (NA1) and C-terminal (P3C) P1 fragments (Fig. 1A), which have been shown to form a stable high-affinity and functionally active complex (17). The NA1/P3C protein complex was copurified and then cocrystallized for X-ray diffraction data collection to 2.0-Å resolution. We observed that the N terminus adopts a previously unidentified fold that serves as an intramolecular scaffold connecting it to C-terminal portions of the molecule, thus locking P1’s hybrid helical stalk into place. We validated this model experimentally using differential scanning calorimetry (DSC) and circular dichroism to demonstrate decreased thermal stability and altered secondary structure in a P1 mutant containing two extra amino acids within the region that normally reacts with the N-terminal intramolecular scaffold. Our X-ray crystallography model and stability measurements agree well with biophysical data characterizing the NA1/P3C complex (17) and provide mechanistic insight into why the N-terminal segment supports the proper folding, function, and stability of the full-length P1 protein. This information contributes to our ability to interpret data regarding protective and nonprotective immune responses and preventative therapies, and will inform future studies evaluating bacterial adhesion and biofilm formation by S. mutans and related organisms.     

From the Cover: Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria

Successful diagnosis, screening, and elimination of malaria critically depend on rapid and sensitive detection of this dangerous infection, preferably transdermally and without sophisticated reagents or blood drawing. Such diagnostic methods are not currently available. Here we show that the high optical absorbance and nanosize of endogenous heme nanoparticles called “hemozoin,” a unique component of all blood-stage malaria parasites, generates a transient vapor nanobubble around hemozoin in response to a short and safe near-infrared picosecond laser pulse. The acoustic signals of these malaria-specific nanobubbles provided transdermal noninvasive and rapid detection of a malaria infection as low as 0.00034% in animals without using any reagents or drawing blood. These on-demand transient events have no analogs among current malaria markers and probes, can detect and screen malaria in seconds, and can be realized as a compact, easy-to-use, inexpensive, and safe field technology.Rapid, accurate, noninvasive, and bloodless detection of low levels of malaria parasites is critical for surveillance, treatment, and elimination of malaria (1, 2) but so far is not supported by current diagnostic methods (3–7), which depend upon qualified personnel, sophisticated in vitro methodologies, blood sampling, and specific reagents. All blood-stage malaria parasites digest hemoglobin and form unique intraparasite nanoparticles called “hemozoin” (8–11). The high optical absorbance combined with the nanosize (50–400 nm) of hemozoin (9, 11, 12) can be used to generate a transient localized vapor nanobubble around hemozoin in response to a short, safe laser pulse. A short picosecond pulse localizes the released heat to a nanovolume around a nanoparticle (13, 14) and evaporates liquid around the hemozoin in an explosive manner, creating an expanding and collapsing transient vapor bubble of submicrometer size in the malaria parasite (Fig. 1A). Using our experience in the generation and detection of vapor nanobubbles of other origins in cells and animals (15, 16), we hypothesized that hemozoin-induced vapor nanobubbles (H-VNBs) can act as highly sensitive optical and acoustic probes for malaria detection (Fig. 1 B and C). Here, we report studies of H-VNBs in water, whole blood, and individual human red blood cells (RBCs) infected with Plasmodium falciparum and evaluate their noninvasive transdermal detection in Plasmodium yoelii–infected mice.Open in a separate windowFig. 1.(A) Laser-induced generation of a transient H-VNB around hemozoin nanoparticles inside a malaria parasite within the iRBC. Detection of optical scattering signals of the nanobubble with an optical detector (OD) (B) and (C) pressure pulse of the H-VNB detected with an ultrasound transducer (UT) as an acoustic trace.     

Functional conservation despite structural divergence in ligand-responsive RNA switches

An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.Internal ribosome entry site (IRES) elements provide an alternative mechanism for translation initiation by directing the assembly of functional ribosomes directly at the start codon in a process that does not require 5′ cap recognition or ribosomal scanning and that is independent of many host initiation factors (1–4). The genomes of Flaviviridae and Picornaviridae contain elements that share similarity with the archetypical hepatitis C virus (HCV) IRES in overall domain organization, but not sequence or details of secondary structure (5). The HCV IRES adopts a complex architecture of four independently folding domains (Fig. 1A) (6). Domain II is nearly 100% conserved in clinical isolates (7) and has analogous counterparts in other viral IRES elements, all of which display some secondary structure similarity, but significant sequence variation in the subdomain IIa-like internal loop (Fig. 1B). Domain II has been shown to promote stable entry of HCV and classic swine fever virus (CSFV) mRNA at the decoding groove of the 40S subunit (8–10) and is required for initiation factor removal before ribosomal subunit joining (11), as well as adjustment of initiator tRNA orientation (12). The transition from initiation to elongation stages of translation depends critically on domain II (13). Recently, direct interaction of HCV domain II with initiator tRNA has been demonstrated (14). In HCV, subdomain IIa folds into an L-shaped motif (15) (Fig. 1C) that introduces a 90° bend in domain II (16) and directs the IIb hairpin toward the E-site at the ribosomal subunit interface (17, 18).Open in a separate windowFig. 1.Structures and ligands of viral IRES. (A) The IRES in the 5′ UTR of the HCV genome. The location of subdomain IIa is highlighted by an orange box. The viral genome encodes structural (S) and nonstructural (NS) proteins and contains a structured 3′ UTR. (B) Secondary structure predictions of domain II motifs in viral IRES elements from HCV and other flaviviruses, including CSFV and BVDV, as well as picornaviruses such as AEV and SVV. Non-Watson–Crick base pairs are indicated by the ○ symbol. Sequence conservation is indicated in red. (C) Crystal structure of the subdomain IIa RNA from HCV. (D) Benzimidazole (1) and diaminopiperidine (2) inhibitors of IRES-driven translation that target the HCV subdomain IIa. (E) Crystal structure of the HCV subdomain IIa RNA in complex with inhibitor 1.The HCV IRES subdomain IIa is the target for viral translation inhibitors (Fig. 1D) that bind to the internal loop and block translation by capturing distinct conformational states of the RNA (7). Structure analysis revealed that benzimidazole inhibitors such as compound 1 (19, 20) interact with an extended architecture of IIa in which the stems flanking the internal loop are coaxially stacked on both sides of the ligand-binding pocket (Fig. 1E) (21). In contrast, diaminopiperidine compounds such as 2 bind and lock the IIa RNA in a bent conformation that corresponds to the ligand-free state (22). Conformational capture of the subdomain IIa switch by ligands in solution was demonstrated by FRET experiments and established as a mechanism of IRES inhibition (23). On the basis of these findings, it was proposed that subdomain IIa may be the target for a cognate biological ligand whose adaptive recognition by the RNA motif may facilitate ribosome release from the IRES-bound complex (7).Here, we have explored potential candidates for a cognate ligand of the subdomain IIa switch and investigated the structural and functional conservation of similar ligand responsive switch motifs in other IRES RNAs.     

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169–172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.Increasing demand for fuels, geopolitical instability, the limitation of global petroleum reserves, and the impact on climate change induced by greenhouse gases have increased the need for renewable and sustainable biofuels (1–5). First-generation biofuels produced from food crops, such as corn, are limited by cost and competition with food supply (6, 7). Switchgrass is a perennial grass native to North America, and its high productivity on marginal farmlands and low agricultural input requirements make it an attractive feedstock for the production of biofuels and biochemicals (8). A yield of 36.7 Mg⋅ha⁻¹ was achieved in field trials in Oklahoma (9), and switchgrass has the potential to produce 500% or more energy than is used for its cultivation (10). The use of abundant lignocellulosic plant biomass as feedstock is environmentally desirable and economically essential for enabling a viable biofuels industry (11). Current strategies for bioethanol production from lignocellulosic feedstocks require three major operational steps: physicochemical pretreatment, enzymatic saccharification, and fermentation (Fig. 1) (6, 12). Pretreatment and enzymatic hydrolysis represent substantial cost and it is estimated that the use of cellulolytic microbes for consolidated bioprocessing and eliminating pretreatment would reduce bioprocessing costs by 40% (2). Considerable effort has been made to develop single microbes capable of both saccharification and fermentation to avoid the substantial expense of using saccharolytic enzyme mixtures (13). Heterologous expression of saccharolytic enzymes has been demonstrated in a number of organisms, including Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli, and Bacillus subtilis to ferment various model cellulosic and hemicellulosic substrates (13–15). Although these approaches have resulted in progress in cellulose utilization, the overall enzyme activity is still very low compared with that of naturally cellulolytic organisms and the rates of hydrolysis are not sufficient for an industrial process (13).Open in a separate windowFig. 1.Comparison of bioethanol production strategies and a predicted fermentative pathway in C. bescii. Depiction of “single step bioprocessing” accomplished by engineered C. bescii. CBP, consolidated bioprocessing.High-temperature fermentations facilitate biomass deconstruction and may reduce contamination and volatilize toxic products, such as alcohols. Clostridium thermocellum and Thermoanaerobacterium saccharolyticum have been used in mixed culture fermentations successfully for laboratory-scale demonstration of first-generation consolidated bioprocessing (13, 16) (Fig. 1). C. thermocellum is one promising candidate for consolidated bioprocessing because it is naturally cellulolytic, able to hydrolyze cellulose at 2.5 g⋅L⁻¹⋅h⁻¹, and produces ethanol as one fermentation product, but it has not yet been engineered to produce ethanol at high yield and lacks the ability to ferment hemicellulosic sugars (13, 17). Caldicellulosiruptor bescii, on the other hand, is the most thermophilic cellulolytic bacterium so far described, growing optimally at ∼80 °C with the ability to use a wide range of substrates, such as cellulose, hemicellulose, and lignocellulosic plant biomass without harsh and expensive chemical pretreatment (17, 18), efficiently fermenting both C₅ and C₆ sugars derived from plant biomass (17, 18). C. bescii uses the Embden–Meyerhof–Parnas pathway for conversion of glucose to pyruvate, and the predominant end-products are acetate, lactate, and hydrogen (Fig. 2) (18). A mutant strain of C. bescii (JWCB018) was recently isolated in which the lactate dehydrogenase gene (ldh) was disrupted spontaneously via insertion of a native transposon (19, 20). A complete deletion of ldh was also engineered (21), and this strain no longer produced lactate, instead diverting metabolic flux to additional acetate and H₂, demonstrating the utility of the newly developed tools to provide a platform for further strain engineering. The recent development of genetic methods for the manipulation of this organism (19, 21, 22) opens the door for metabolic engineering for the direct conversion of unpretreated plant biomass to liquid fuels, such as ethanol, via “single step bioprocessing” (Fig. 1).Open in a separate windowFig. 2.Overview of C. bescii fermentative pathways for bioconversion of hexose sugars. Pathway 1 (blue) results in 2 mol of acetic acid and 4 mol of H₂ per mole of glucose. Pathway 2 (green) produces 2 mol of lactic acid per mole of glucose. Pathway 3 (red) is a new pathway resulting from heterologous expression of the C. thermocellum adhE gene to synthesize 2 mol of ethanol per mole of glucose.     

XerD-mediated FtsK-independent integration of TLC? into the Vibrio cholerae genome

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXϕ, the cholera toxin phage. Here, we show that in contrast, integration of TLCϕ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXϕ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCϕ, along with any CTXϕ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXϕ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.The causative agent of the epidemic severe diarrheal disease cholera is the Vibrio cholerae bacterium. A major determinant of its pathogenicity, the cholera enterotoxin, is encoded in the genome of the filamentous cholera toxin phage, CTXϕ (1). Like many other V. cholerae filamentous phages, CTXϕ uses a host chromosomally encoded, site-specific recombination (Xer) machinery for lysogenic conversion (2–4). The Xer machinery normally serves to resolve chromosome dimers, which result from homologous recombination events between the two chromatids of circular chromosomes during or after replication. In V. cholerae, as in most bacteria, the Xer machinery consists of two tyrosine recombinases, XerC and XerD. They act at a unique specific chromosomal site, dif, on each of the two circular chromosomes, chrI and chrII, of the bacterium (5). Integrative mobile elements exploiting Xer (IMEXs) carry a dif-like site on their circular genome, attP (3, 4) (Fig. 1A). XerC and XerD promote their integration by catalyzing a recombination event between this site and a cognate chromosomal dif site (3, 4) (Fig. 1A). Based on the structure of their attP site, IMEXs can be grouped into at least three families (3, 4) (Fig. 1B). In all cases, however, a new functional dif site is restored after integration, which permits multiple successive integration events (Fig. 1A). Indeed, most clinical and environmental V. cholerae isolates harbor large IMEX arrays (6, 7).Open in a separate windowFig. 1.Systems that use Xer. (A) Scheme depicting the sequential integration of IMEXs. Triangles represent attP and dif sites, pointing from the XerD binding site to the XerC binding site. Chromosomal DNA (black), TLCϕ DNA (blue), and CTXϕ DNA (magenta) are indicated. Dotted triangles represent nonfunctional CTXϕ sites. (B) Sequence alignment of dif1, attP^CTX, attP^VGJ, attP^TLC, difA, and dif2. Bases differing from dif1 are indicated in color. Bases that do not fit the XerD binding site consensus are indicated in lowercase. XerC (●) and XerD (○) cleavage points are indicated. (C) Xer recombination pathways. XerC (light gray circles), XerD (dark gray circles), dif sites (red and black lines), and attP^CTX and attP^VGJ (magenta and green lines) are indicated. XerC and XerD catalysis-suitable conformations are depicted as horizontal and vertical synapses, respectively. Cleavage points are indicated as in B.IMEX array formation participates in the continuous and rapid dissemination of new cholera toxin variants in at least three ways. First, CTXϕ integration is intrinsically irreversible because the active form of its attP site consists of the stem of a hairpin of its ssDNA genome, which is masked in the host dsDNA genome (8, 9) (Fig. 1 A and B). However, free CTXϕ genome copies can be produced by a process analogous to rolling circle replication after the integration of a second IMEX harboring the same integration/replication machinery, such as the RS1 satellite phage, which permits the production of new CTXϕ viral particles (10). Second, the V. cholerae Gillermo Javier filamentous phage (VGJϕ) belongs to a second category of IMEXs whose attP site permits cycles of integration and excision by Xer recombination (11). VGJϕ excision allows for the formation of hybrid molecules harboring the concatenated genomes of CTXϕ and VGJϕ, provided that VGJϕ integrated before CTXϕ (11). The hybrid molecules can be packaged into VGJϕ particles. VGJϕ particles have a different receptor than CTXϕ, which permits transduction of the cholera toxin genes to cells that do not express the receptor of CTXϕ (11–13). Finally, integration of the toxin-linked cryptic phage (TLCϕ), a satellite phage that defines a third category of IMEXs, seems to be a prerequisite to the toxigenic conversion of many V. cholerae strains (14, 15). IMEXs from this family are found integrated in the genome of many bacteria outside of the Vibrios, including human, animal, and plant pathogens, which sparked considerable interest in the understanding of how they exploit the Xer machinery at the molecular level (3, 4).Xer recombination sites consist of 11-bp XerC and XerD binding arms, separated by an overlap region at the border of which recombination occurs (Fig. 1B). XerC and XerD each promote the exchange of a specific pair of strands (Fig. 1B). Recombination between dif sites is under the control of a cell division protein, FtsK, which restricts it temporally to the time of constriction and spatially to a specific zone within the terminus region of chromosomes (16–19). FtsK triggers the formation of a Holliday junction (HJ) by XerD catalysis, which is converted into product by XerC catalysis after isomerization (20, 21) (Fig. 1C). The intermediate HJ is stable enough to be converted into product by replication when XerC catalysis is impeded (5, 17) (Fig. 1C). The integration of IMEXs of the CTXϕ and VGJϕ families escapes FtsK control. The lack of homology in the overlap regions of their attP sites and the dif sites they target prevents any potential XerD-mediated strand exchange (Fig. 1B). CTXϕ and VGJϕ rely on the exchange of a single pair of strands by XerC catalysis for integration, with the resulting HJ being converted into product by replication (8, 9, 11) (Fig. 1C). In the case of CTXϕ, integration is facilitated by an additional host factor, EndoIII, which impedes futile cycles of XerC catalysis once the pseudo-HJ is formed (22) (Fig. 1C). In contrast, the overlap region of TLCϕ attP, attP^TLC, is fully homologous to the overlaps of dif1 and difA, the two sites in which it was found to be integrated (Fig. 1B). Four integration pathways could thus be considered, depending on whether recombination is initiated by XerC or XerD catalysis, and whether it ends with a second pair of strand exchange or not. In addition, attP^TLC lacks a consensus XerD binding site, which could affect the whole recombination process (Fig. 1B).Here, we show that attP^TLC is a poor XerD binding substrate. Nevertheless, we show that TLCϕ integration is initiated by XerD catalysis and that the resulting HJ is converted into product by XerC catalysis. We further show that TLCϕ integration is independent of FtsK. Finally, we demonstrate that the same reaction can lead to the excision of TLCϕ–CTXϕ arrays, providing a plausible mechanism for how all of the CTXϕ copies integrated on V. cholerae chrI can be eliminated in a single step, as occurred in ancestors of strains from the second wave of the current cholera pandemic (23–25).