Topological constraints and modular structure in the folding and functional motions of GlpG,an intramembrane protease

We investigate the folding of GlpG, an intramembrane protease, using perfectly funneled structure-based models that implicitly account for the absence or presence of the membrane. These two models are used to describe, respectively, folding in detergent micelles and folding within a bilayer, which effectively constrains GlpG''s topology in unfolded and partially folded states. Structural free-energy landscape analysis shows that although the presence of multiple folding pathways is an intrinsic property of GlpG’s modular functional architecture, the large entropic cost of organizing helical bundles in the absence of the constraining bilayer leads to pathways that backtrack (i.e., local unfolding of previously folded substructures is required when moving from the unfolded to the folded state along the minimum free-energy pathway). This backtracking explains the experimental observation of thermodynamically destabilizing mutations that accelerate GlpG’s folding in detergent micelles. In contrast, backtracking is absent from the model when folding is constrained within a bilayer, the environment in which GlpG has evolved to fold. We also characterize a near-native state with a highly mobile transmembrane helix 5 (TM5) that is significantly populated under folding conditions when GlpG is embedded in a bilayer. Unbinding of TM5 from the rest of the structure exposes GlpG’s active site, consistent with studies of the catalytic mechanism of GlpG that suggest that TM5 serves as a substrate gate to the active site.GlpG is a rhomboid protease that sits and functions in the cell membrane. GlpG’s homologs are found across all kingdoms of life. GlpG has been the subject of several biophysical experimental studies aimed toward understanding membrane protein folding and the relationships among protein structure, dynamics, and function (1–5). An extensive experimental φ-value analysis found φ-values significantly different from zero, indicative of structural changes during the rate-limiting step of folding, in transmembrane helices 1 through 5 (TM1-5) and the intervening loops (4). Most of the nonzero φ-values, particularly in TM3-5 and in the large loop L1, were negative, meaning that although the corresponding mutation destabilizes the native state, the mutation nonetheless accelerates folding. The preponderance of negative φ-values was puzzling and unprecedented, and at the time, these effects were tentatively ascribed to nonnative interactions in the transition state ensemble. In this work, we show that, in fact, simple models with perfectly funneled energy landscapes that lack nonnative interactions are able to explain the origin of these negative φ-values and how the values arise when folding in detergent micelles rather than bilayers.α-Helical membrane protein folding is thought to occur in two stages in vivo (6). The first stage, setting up the proper topology of transmembrane helices, is handled by the translocon (7, 8). In the present context, topology refers to specifying the directions in which a membrane protein’s constituent transmembrane helices traverse the bilayer. The second stage, converting from properly inserted but dissociated helices into a functional folded structure, occurs spontaneously and is, in some ways, analogous to soluble protein folding. However, we know, ranging from the hydrophobic effect (9, 10) to water-mediated (11) and screened electrostatic interactions (12), the solvent plays a role in determining what types of noncovalent interactions are stabilizing and destabilizing. Whereas soluble proteins fold in polar and isotropic aqueous solutions, membrane proteins fold in largely apolar and anisotropic environments. These environmental differences complicate applying directly methods developed for studying soluble protein folding to the study of membrane protein folding. Nonetheless, experimentalists have been able to apply a variety of methods to study the kinetics and thermodynamics of membrane protein folding through the use of detergent micelles as a membrane-mimicking environment. Experiments that probe the folding mechanisms of membrane proteins have used micelles composed of a mixture of anionic and nonionic detergents (4, 13, 14), which not only keep membrane proteins soluble but also, through use of mixed micelles, allow the equilibrium between folded and unfolded states to be tuned. Micelles predominantly composed of nonionic detergents, such as n-dodecyl-β-d-maltopyranoside (DDM), preferentially stabilize a folded state that has been shown to be functional and is therefore likely to be structurally similar to the folded state in vivo. Micelles predominantly composed of anionic detergents, on the other hand, preferentially stabilize an unfolded state that contains significant amounts of secondary structure. This ability to tune the equilibrium means that stopped-flow kinetic experiments can be combined with protein-engineering techniques to determine folding mechanisms at the single-residue level (4, 13, 15), in analogy to what has been done for soluble proteins (16–18). Because carrying out these types of experiments in bilayers is still difficult, it is presently unknown how folding mechanisms determined in micelles compare with those in membranes. Confining proteins to a 2D membrane is expected to constrain unfolded and partially folded ensembles to having structures with helices that are largely properly aligned and embedded in the membrane; such topological restrictions would be relaxed in a micellar environment.Theoretical (19, 20) and experimental (3, 4) work suggests that at least some membrane proteins can reversibly fold and unfold without the aid of the translocon or chaperones in vitro. It is therefore likely that membrane protein folding landscapes are funneled, much like globular protein landscapes (21, 22). Structure-based models with perfectly funneled energy landscapes have proven useful for investigating the folding and binding of proteins (23, 24). In this study, we use a structure-based model to investigate folding of a membrane protein in two different situations: in the absence and presence of an implicit membrane energy term that biases conformations to have the correct topology with respect to the membrane. Simulations with the implicit membrane term are thus taken to model folding in a bilayer, whereas simulations without the implicit membrane energy are taken to model folding in detergent micelles. Although this way of modeling micelles and bilayers is an oversimplification, it captures the significantly increased topological freedom of membrane proteins in micellar environments compared with lipid bilayer. Fig. 1 shows schematic representations of the corresponding denatured states of membrane proteins in bilayers and micelles.Open in a separate windowFig. 1.Schematic diagrams of the unfolded state of α-helical membrane proteins in bilayers (Left) and detergent micelles (Right). The transmembrane helices (cylinders) are connected by loops. Transmembrane helices are either embedded in a membrane (rectangular prism) or are surrounded by detergent micelles (transparent gray spheres). In this work, we use an implicit membrane model to simulate folding within a bilayer and assume that folding in detergent micelles corresponds to folding without constraints on the alignment of helices. In both cases, we assume that the unfolded state has near-native levels of secondary structure, as has been observed in experiments on the SDS-denatured state of membrane proteins.The same energy landscape that dictates folding routes also encodes functional motions. It has been suggested that the modularity in the structure of GlpG supports functional motions (1, 25). The N-terminal domain, which contains transmembrane helices 1 and 2 (TM1-2) as well as the intervening L1 loop, functions as a structural scaffold (25), whereas the C-terminal domain with its four transmembrane helices (TM3-6) includes the catalytic site (25). The C-terminal domain is apparently more flexible than the N-terminal domain; both the loop L5 (5) and the transmembrane helix TM5 (25) have been crystallized in multiple conformations. Because of this flexibility, it has been suggested that either L5 alone (5) or L5 and TM5 (25) may serve as a substrate gate for access to the catalytic site. Using free-energy landscape analysis and perturbation methods along with structural analysis, we show that there is a near-native state significantly populated under folding conditions and elucidate the state’s connections to GlpG’s folding mechanism and function.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Understanding the kinetic mechanism of RNA single base pair formation

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.RNAs perform critical cellular functions at the level of gene expression and regulation (1–4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7, 8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9–19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19–36). However, due to the complexity of the RNA folding energy landscape (37–46) and the limitations of experimental tools (47–55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18, 19, 54–73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on- and off-pathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, base pairing and stacking interactions are broken concomitantly, whereas in the other pathway, base stacking is broken after base pairing is disrupted. Furthermore, the unfolding requires that the Cyt diffuse away from the pairing Gua to a distance such that the C-G hydrogen bond cannot reform easily. More recently, Colizzi and Bussi (75) performed molecular dynamics (MD) pulling simulations for an RNA duplex and construct free energy landscape from the pulling simulation. The simulation showed that the base pair opening reaction starts with the unbinding of the 5′-base, followed by the unbinding of the 3′-base (i.e., the 5′-base is less stable than the 3′-base). These previous unfolding simulations offered significant insights into the pathways and transition states. However, as shown below, several important issues remain.One intriguing problem is the rate model for base pairing. There are currently three main types of models. In the first type of model, the barrier ΔG+‡ for closing a base pair is dominated by the entropic cost ΔS for positioning the nucleotides to the base-paired configuration and the barrier ΔG−‡ for opening a base pair is the enthalpic cost ΔH for disrupting the hydrogen bonds and base stacking interactions (18, 59, 60). In the second type of model, ΔG+‡ is the net free energy change for base pairing ΔG = ΔH ? TΔS and ΔG−‡ is zero (76, 77). In the third type of model, ΔG±‡=±ΔG/2 is used (78). In addition to the above three main types, other models, such as more sophisticated hybrid rate models, have been proposed (29).In this paper, we report a hybrid method (see Fig. 1) to investigate the single base pairing process. In contrast to the previous simulations for temperature- or force-induced unfolding reactions, we directly model the folding process here (i.e., the base pair closing process). Specifically, we use MD simulations to identify the conformational clusters. Based on the network of the conformational clusters as a reduced conformational ensemble, we apply kinetic Monte Carlo (KMC) and master equation (ME) methods to elucidate the detailed roles of base pairing and stacking interactions, as well as the roles of water and ions (79–82). The study reveals previously unidentified kinetics pathways, misfolded states, and rate-limiting steps. A clear understanding of the microscopic details of the elementary kinetic move is a prerequisite for further rigorous study of large-scale RNA kinetic studies. The method described here may provide a feasible way to develop a rate model for the base pair/stack-based kinetic move set. Furthermore, the mechanism of RNA single base folding may provide useful insights into many biologically significant processes, such as nucleotide flipping (83) in helicases and base pair fraying (84) (as the possible first step for nucleic duplex melting in nucleic acid enzymatic processes).Open in a separate windowFig. 1.(A) Folding of a single nucleotide (G1, red) from the unfolded (Left) to the native folded (Right) state. (B) Exhaustive sampling for the (discrete) conformations of the G1 nucleotide (Right) through enumeration of the torsion angles (formed by the blue bonds). (C) Schematic plot shows the trajectories on the energy landscape (depicted with two reaction coordinates for clarity) explored by the MD simulations. The lines, open circles, and hexagons denote the trajectories; the initial states; and the (centroid structures of the) clusters, respectively. (D) Conformational network based on six clusters. (E) The rmsds to the different clusters provide information about the structural changes in a MD trajectory.     

Designed protein reveals structural determinants of extreme kinetic stability

The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.The design of proteins with a desired stable fold and function is a much sought after goal. Although impressive recent successes have been reported in designing both natural and novel protein functions and/or structures (1–6), design remains difficult, often requiring multiple rounds of iterative improvements (7–10). In depth biophysical characterization of protein design outcomes and an understanding of their molecular basis have been limited, and these are critical for improving future designs. Combining designed function with structure is particularly difficult, in part because functional sites tend to be sources of thermodynamic instability (11, 12) and folding frustration (13–15). We investigate how an approach that considers both structure and function from the outset may be used to overcome such obstacles. Furthermore, we demonstrate how kinetic and related stabilities against denaturation can be rationally designed.A promising emerging paradigm for protein design is the repetition of modular structural elements (1, 2, 5–7, 14, 16–20). This approach can simplify the design process and build on aspects of the evolution of natural repetition in proteins, as well as incorporate the inherent multivalent binding functionality of such structures (1, 21). Internal structural symmetry, resulting from the repetition of smaller elements of structure, is very common in natural proteins, with ∼20% of all protein folds (22) and the majority of the most populated globular protein folds (superfolds) (21) containing internal structural symmetry. Recent design successes, for helical proteins (5, 6), repeat proteins (18, 20, 23) and symmetric superfolds (1, 2, 7, 16, 17, 19, 24–26) recommend the simplification of the design process by using repetitive/symmetric folds as a particularly effective strategy.The β-trefoil superfold is an interesting test case for design by repetition as bioinformatics analysis has revealed multiple and recent instances of the evolution of distinct proteins with this symmetric fold (1). The fold consists of three repeats, each containing four β-strands, and is adopted by numerous superfamilies with highly diverse binding functions (27). Our design of a completely symmetric β-trefoil, ThreeFoil (Fig. 1), used a hypothetical multivalent carbohydrate binding template and mutated 40 of the 141 residues (1). The mutations were based on a combination of consensus design using a limited set of close homologs (to preserve function), and energy scoring using Rosetta (28). The design was successful on the first attempt, producing a soluble, well folded, and functional monomer with very high resistance to structural fluctuations as indicated by high resistance to thermal denaturation and limited amide H/D exchange (1).Open in a separate windowFig. 1.Design of ThreeFoil. (A) ThreeFoil (PDB: 3PG0) illustrating its three identical peptide subdomains (red, green, blue). (B) ThreeFoil’s secondary structure: turn (purple), β-strand/bridge (yellow), and 3/10-helix (magenta) and ligand binding residues indicated by colored circles and insertions shown in red. (C) Comparison of ThreeFoil with the independently designed Symfoil (PDB: 3O4D, 15% sequence identity), shown along (Left) and across (Right) the axis of symmetry. Backbones are colored by RMSD between the two structures (blue to white, 0–5 Å), with insertions in the loops of ThreeFoil relative to Symfoil colored red. ThreeFoil’s bound sodium shown in gray, and bis-Tris, which binds in the conserved carbohydrate binding sites, shown in cyan.Here, we use a battery of biophysical and computational methods to perform an in depth analysis of Threefoil, which shows that it has remarkably slow unfolding and folding kinetics compared with natural and designed proteins due to an unusually high transition state energy barrier. Such kinetic stability against unfolding has been studied little to date. Furthermore, Threefoil is extremely resistant to chemical denaturation and proteolytic degradation. Analyses using Absolute Contact Order (ACO) (29) and Long-Range Order (LRO) (30) as well as Gō model folding simulations (31–33) show that ThreeFoil’s resistance can be explained by the high cooperativity of its folded structure, which includes many long-range interactions. Simulations also show that nonnative interactions or folding frustration arising from protein symmetry (34) do not create long-lived traps during folding or account for the high barrier. They also explain how ligand binding can chaperone folding, which can be an added advantage of designing the fold and function together. Notably, additional analyses using whole proteome screening and other experiments show that proteins with similar resistances as ThreeFoil generally have high ACO/LRO values. Thus, the design method used for ThreeFoil and the strategy of designing folds with many long-range contacts may be useful for designing functional proteins with high resistance to denaturation and degradation, as may be needed for challenging biotechnology applications.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein

Protein–protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.For many proteins, correct folding to a specific 3D structure is essential for their function inside the cell; once folded, some of these have the appropriate shape and accessible chemical groups to interact specifically with, and bind to, another protein (1). However, for a number of protein–protein interactions, folding and binding do not appear to be separate, sequential events (2, 3). Many intrinsically disordered proteins (IDPs) will appear largely unfolded in isolation, only forming a specific structure when bound to an appropriate partner protein and undergoing coupled folding and binding (4–6). Such reactions are abundant in signaling and regulatory processes (7, 8). Protein folding does not simply provide correctly shaped building blocks for the cell; it can play an intimate role in molecular recognition.Over the past decade, bioinformatics studies have revealed that protein disorder (7, 9), and coupled folding and binding (10), are widespread in biology. Many structures of bound, folded IDPs have been solved and have shown the wide range of topologies that can be formed (11). Biophysical techniques (12), NMR in particular (13), can characterize isolated IDPs in detail. Despite this progress, the number of studies examining kinetics and the mechanisms of binding remains relatively small (14–21) given that the most commonly observed function of IDPs is in coupled folding and binding reactions (22).To describe coupled folding and binding, two extreme mechanisms are often discussed, focusing on whether an IDP needs to fold before interacting productively with its binding partner. In isolation an IDP could, perhaps only transiently, occupy a conformation that resembles the bound state. In the pure conformational selection mechanism, the IDP must be in this conformation at the start of the eventually successful encounter with the partner protein (23, 24) (Fig. 1A). Arguments in support of this mechanism largely come from NMR studies that have successfully detected these lowly populated, folded states in unbound IDPs (25–27). In the contrasting induced-fit mechanism, there is no requirement for the IDP to fold in isolation (28). Instead, the potentially transient interactions with the partner protein lead to the folding of the IDP (Fig. 1A). Complex mixtures of these two extreme mechanisms can also be imagined: e.g., perhaps only a proportion of the IDP needs to fold before the encounter, i.e., conformational selection followed by induced fit of the remaining peptide chain (29). To add to the potential complexity, flux through different pathways could occur simultaneously, and may depend on the concentrations of protein involved (23, 30). Further, confirming the degree of induced fit and conformational selection is only one aspect of the binding mechanism. There remain a large number of mechanistic possibilities beyond the state of the IDP prior to successful encounters.Open in a separate windowFig. 1.PUMA–MCL-1 binding. (A) Cartoon of binding mechanisms. IDP PUMA (blue) can undergo coupled folding and binding with structured MCL-1 (white) to form a single, contiguous α-helix. Structures based on PDB 2ROC (39) and 1WSX (58). Unbound PUMA and encounter complex built using Chimera (University of California, San Francisco). Figure prepared using PyMol. (B) Representative fluorescence stopped-flow traces for binding. Increasing the concentration of urea from 0 to 3.5 M (in 0.5-M increments) slows association. (C) The urea dependence of the natural log of the association rate constant (k+) for the wild-type PUMA peptide used in this study. (D) The urea dependence of the dissociation rate constant (k−). k− was determined by preforming the PUMA–MCL-1 complex at micromolar concentrations and manually diluting to nanomolar concentrations to induce dissociation. The resulting kinetic trace was fit to a reversible model, fixing k+ from the association experiments (41). Gradient of the linear fits corresponds to the m values discussed in the main text. A, B, and C adapted from ref. 37.It is largely agreed that most protein folding (and unfolding) reactions are limited by the requirement to populate a high-energy transition state (31). Kinetic, time-resolved experiments, in combination with site-directed mutagenesis and Φ-value analysis (32), have been applied successfully to describe these transition states (33, 34). With carefully chosen mutations, the distribution of Φ values (classically between 0 and 1) offers an average picture of the interactions formed at this critical stage of the folding reaction, at residue-level resolution. This picture, in conjunction with other evidence, can offer invaluable insights into the mechanisms of folding (35, 36).We have previously reported the kinetics of a model coupled folding and binding reaction (37, 38); the BH3 motif of PUMA (an IDP) can associate with the structured protein MCL-1 and fold to a single contiguous α-helix (39). The solvent and temperature dependence of the association reaction suggested that this reaction is limited by a free energy barrier, or transition state (TS) (37). Here we systematically make structurally conservative mutations to the IDP and the partner protein, apply Φ-value analysis, and describe the transition state for binding. Molecular dynamics simulations using a coarse-grained, topology-based model of the binding process are consistent with our experimental results. We bring together all available evidence to propose a mechanism of binding.     

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.     

Energy landscape views for interplays among folding,binding, and allostery of calmodulin domains

Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, “combined spontaneous folding and induced fit” occurs, whereas at a higher concentration, “binding-induced folding” dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.Protein folding and conformational dynamics have often been characterized by the energy landscape of proteins (1–5). The energy landscape is dependent on the molecular physiochemistry and thus is modulated by many factors, such as chemical modification and ligand binding. Ligand binding, in turn, is dependent on the conformation of proteins. Thus, folding, binding, and allosteric conformational dynamics are mutually correlated. Despite their obvious correlation in concept, it has been very challenging to characterize how they are indeed coupled for any single proteins. Here, we address, in depth, how these three types of dynamics, folding, binding, and allosteric conformational dynamics, are coupled from the energy landscape perspective for a specific protein, calmodulin (CaM).CaM is a ubiquitous calcium-binding messenger protein involved in signal transduction (6) and, more importantly here, has been a model protein to investigate folding, binding, and allostery. Full-length CaM has two nearly symmetric globular domains connected by a flexible central helix (7, 8). Each domain is composed of paired EF hands containing two Ca²⁺-binding sites (Fig. 1A). Upon binding to Ca²⁺, each CaM domain undergoes substantial conformational change from a closed state to an open state, exposing a hydrophobic patch that can bind with target proteins and regulate downstream processes (9). CaM has been frequently used as a model in studying the folding of multidomain proteins (10, 11), allosteric transitions (12–14), slow conformational dynamics around physiological temperatures (15–18), metal ion binding (19, 20), and correlation between inherent flexibility and protein functions (21, 22). For example, using structure-based coarse-grained (CG) simulations, Chen and Hummer elucidated the coexistence of an unfolded state, a closed state, and an open state around physiological temperatures for the C-terminal domain of CaM (CaM-C) without Ca²⁺ binding (15), which reconciles some seemingly contradictory experimental observations on the slow conformational dynamics of CaM.Open in a separate windowFig. 1.(A) Three-dimensional structure of calmodulin domain at closed [Protein Data Bank (PDB) code: 1cfd] and open states (PDB code:1cll). Calcium ions are represented by yellow spheres. (B) Schematic of coupling among folding, calcium binding, and allosteric motions for the CaM domain. Due to the conformational transitions between open and closed states, in addition to the direct folding pathway (red solid arrow), folding to the most stable state may involve an alternative pathway via a metastable state (green arrow plus blue arrow). The calcium binding can modulate the relative stability of the conformational states and therefore the population of folding pathways. O, C, and U represent open, closed, and unfolded states, respectively.More recently, Rief and coworkers studied the Ca²⁺-dependent folding of CaM based on a new generation technique of single-molecule force spectroscopy, which can probe the reversible folding/unfolding transitions with near equilibrium conditions (10, 23, 24). Their results revealed that at high Ca²⁺ concentrations, the folding pathway of the CaM domain proceeds via a transition state capable of binding Ca²⁺ ions, demonstrating the coupling between Ca²⁺ binding and CaM folding. All these computational and experimental works provided unprecedented understanding of many aspects of the folding and allosteric transitions of CaM. However, a full picture of the coupling among folding, Ca²⁺ binding, and allosteric motions, as schematically shown in Fig. 1B, is still lacking. Particularly, two fundamental issues arising from the allostery and Ca²⁺-binding characteristics of CaM remain elusive: (i) How does the allosteric feature of the energy landscape contribute to the folding complexity? And (ii) how can the folding mechanism of CaM be modulated by Ca²⁺ binding?Motivated by previous computational and experimental studies (15, 23), in this work we investigated the folding coupled with Ca²⁺ binding and allosteric motions of the isolated CaM domains as well as the full-length CaM. To do so, we first integrated computational tools developed for folding, ligand binding, and allosteric motions together. The proposed CG protein model was used for the subsequent series of molecular dynamics (MD) simulations. First, corresponding to Rief’s experiments, we performed MD simulations of isolated CaM domains with pretensions, which gave consistent results with the experiments and, in addition, provided the direct structural assignment on the experimentally observed states. Second, at a higher temperature, without pretension we performed reversible folding/unfolding simulations for a wide range of Ca²⁺ concentrations. The conformational and ligand-binding energy landscape revealed as many as nine distinctive states. Then, we analyzed the binding-coupled folding reactions in terms of the nine states, finding multiple routes and their modulation by Ca²⁺ concentrations. Interestingly, as the Ca²⁺ concentration increases, the CaM domain folding mechanism switches from “combined spontaneous folding and induced fit” to “binding-induced folding,” which accords with the scenario deduced from single-molecule force spectroscopy experiments. Finally, the effects of tension on the conformational fluctuations of the full-length CaM are discussed.     

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrate-ClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex. We establish that ClpS promotes delivery only in cis, as demonstrated by mixing ClpS variants with distinct substrate specificity and either active or inactive NTE truncations. Importantly, we find that ClpA engagement of the ClpS NTE is crucial for ClpS-mediated substrate delivery by using ClpS variants carrying “blocking” elements that prevent the NTE from entering the pore. These results support models in which enzymatic activity of ClpA actively remodels ClpS to promote substrate transfer, and highlight how ATPase/motor activities of AAA+ proteases can be critical for substrate selection as well as protein degradation.AAA+ molecular machines power cellular processes as diverse as protein degradation, microtubule severing, membrane fusion, and initiation of DNA replication, with the common theme that macromolecules are actively remodeled (1–3). Furthermore, protein quality control in all organisms involves deployment of ATP-dependent proteases, consisting of hexameric AAA+ rings that unfold and translocate specific substrates into an associated peptidase barrel (3, 4). Adaptor proteins are known to aid recognition and degradation of certain substrates (5–8), but how enzyme–adaptor pairs ensure proper substrate selection is poorly understood.In prokaryotes and eukaryotes, the N-end rule pathway governs degradation of proteins with specific N-terminal amino acids (9, 10). In Escherichia coli, the primary destabilizing N-degron amino acids are Phe, Tyr, Trp, and Leu (11, 12). ClpS, a widespread bacterial adaptor, recognizes and delivers N-degron substrates to the ClpAP or ClpCP AAA+ proteases (6, 11, 13). These enzymes consist of the AAA+ ClpA or ClpC unfoldases coaxially stacked with the ClpP peptidase (14–16). In eukaryotes, a family of E3 ligases shares homology with the substrate-binding region of ClpS (17, 18). These ligases recognize N-degron substrates and promote ubiquitination, which then targets the modified protein to the 26S proteasome (17, 18).Multiple crystal structures reveal the regions of ClpS that bind to the N-degron, as well as a patch that binds the N-terminal domain of ClpA (19–22). This bivalent binding to the substrate and the enzyme tethers N-degron substrates to ClpAP. Tethering alone is insufficient for ClpS to promote substrate delivery however, given that deletion of 12 amino acids of the ClpS unstructured N-terminal extension (NTE; residues 1–25 in E. coli ClpS) (Fig. 1A) prevents N-degron substrate degradation, but does not block formation of a high-affinity delivery ternary complex (HADC) consisting of substrate, the ClpS adaptor, and the ClpAP protease (Fig. 1B) (19). Importantly, the identity of the NTE sequence is not critical for ClpS function (23). An active delivery model has been proposed in which the translocation pore of ClpA engages the ClpS NTE with subsequent translocation that remodels the delivery complex to achieve substrate engagement (Fig. 1C) (19).Open in a separate windowFig. 1.Model for the active delivery mechanism used by ClpS. (A) The adaptor protein ClpS has a long, flexible N-terminal extension (NTE; residues 1–25) and a folded core domain (ClpS^core; residues 26–106). ClpS^core binds N-degrons. A substrate Tyr in the binding pocket is shown in red (Protein Data Bank ID code 3O1F). Successful substrate delivery requires that the ClpS NTE be at least 14-aa long (shown in green). (B) Formation of an HADC between ClpS, substrate, and ClpA (19) involves formation of additional contacts among ClpA, ClpS, and the N-degron substrate. Assembly of this complex increases the affinity of the substrate for ClpAS by ∼100-fold (19). (C) The current model for ClpA-driven disassembly of the HADC and N-degron substrate delivery. Translocation-mediated ClpA “pulling” on the NTE remodels the ClpS^core structure, weakens the interactions of ClpS with the N-degron, and facilitates its transfer to a site in the ClpA pore. Finally, because ClpS cannot be unfolded by ClpA (19), the adaptor escapes the enzyme, and the substrate is unfolded by ClpA and subsequently degraded by ClpP.In the present work, we investigated how the ClpS NTE functions during delivery of N-degron substrates. We show that the NTE can only promote delivery of substrates that are bound to the same ClpS molecule. Furthermore, we demonstrate that the NTE enters the ClpA translocation pore and provide strong evidence that ClpA pulls on the ClpS NTE to trigger substrate delivery.     

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.Contraction of heart muscle is initiated by an intracellular Ca²⁺ transient followed by binding of Ca²⁺ to troponin in the thin filaments. The resulting change in thin filament structure allows myosin heads or motor domains from the thick filaments to bind to actin in the thin filaments, and a change in the conformation of the actin-attached head domain linked to ATP hydrolysis drives force generation and filament sliding (1–3). During this “working stroke,” small conformational changes in the catalytic domain of the myosin head associated with release of ATP hydrolysis products are amplified by its light chain domain, containing the essential and regulatory light chains (4), to produce a nanometer-scale displacement at the connection of the head to the thick filament backbone.Contractility of heart muscle is also controlled by multiple posttranslational modifications of both thick and thin filament regulatory proteins, and these changes in filament proteins have been widely implicated in the modulation of cardiac output in health and disease (5–7). In the present study, we focused on phosphorylation of the cardiac isoform of the regulatory light chain (cRLC) as a well-characterized and experimentally accessible example of modification of a thick filament component. cRLC is at the thick filament end of the myosin head, where it joins the coiled–coil tail that forms the thick filament backbone. cRLC is partially phosphorylated in vivo under basal conditions (8–12), and changes in its phosphorylation level are linked to heart disease (8, 11, 13, 14). cRLC mutations associated with hypertrophic cardiomyopathy abolish cRLC phosphorylation in vitro (15), and mice expressing nonphosphorylatable cRLCs show severe cardiac dysfunction (10, 16). In the vertebrate heart, cRLCs are phosphorylated almost exclusively by the cardiac isoform of myosin light chain kinase (cMLCK) (17, 18), and cMLCK gene ablation leads to severe cardiac hypertrophy (19).The mechanisms responsible for the regulation of cardiac function by cRLC phosphorylation are poorly understood at the molecular, cellular, and organ levels. Mechanistic hypotheses at the molecular level have been largely based on studies of smooth and skeletal muscle RLCs, which have similar molecular structures and contain phosphorylatable serines analogous to that (Ser15) in cRLC (4, 20–22). Phosphorylation of the analogous serine in smooth muscle RLC (smRLC) is the primary mechanism of contractile regulation in that tissue. Phosphorylation of skRLC plays a similar role in some invertebrate skeletal muscles (23, 24) and enhances contractility in mammalian skeletal muscle (25).Electron microscopy studies of isolated myosins and thick filaments from all these sources (21, 26, 27) suggest a conserved molecular mechanism in which RLC phosphorylation activates or potentiates contractility by disrupting a compact OFF conformation of myosin in which the myosin heads are folded back on the myosin tail (20, 21, 28) (Fig. 1A). In striated muscles, this folded OFF conformation is linked to the formation of an ordered helical lattice of myosin head domains on the surface of the thick filaments, with the long axis of the head roughly parallel to the filament axis. This surface lattice of myosin heads is stabilized by intermolecular interactions between adjacent myosin molecules and possibly between myosin and two other thick filament components, titin, and myosin-binding protein C (MyBP-C). This characteristic folded OFF state of myosin is also present in the C-zone of cardiac thick filaments: the region that contains MyBP-C (26, 27). Phosphorylation of skRLC in isolated thick filaments of skeletal muscle disrupts the lattice, releasing the myosin heads from the filament surface (Fig. 1B). These structural studies on isolated proteins and filaments led to the hypothesis that RLC phosphorylation potentiates contractility in skeletal and cardiac muscle by increasing the number of myosin heads available for actin interaction.Open in a separate windowFig. 1.OFF and ON conformations of the thick and thin filaments in heart muscle. (A) OFF conformations at low [Ca²⁺] in the absence of phosphorylation of the cRLC (blue). (B) ON conformations with calcium (green) bound to troponin C (pink/red) and cRLC phosphorylated (yellow circle, P). Troponin I, troponin T, and tropomyosin are yellow, blue, and sepia, respectively. Essential light chain is brown. BC-cRLC probe dipole is indicated by red double arrows.The aim of the present work was to understand the role of interactions between thick and thin filament-based regulatory mechanisms in a heart muscle cell, in which the normal structure, organization, and interrelationship of the filaments is preserved. We measured the structural changes in the thick filaments of ventricular trabeculae by polarized fluorescence from bifunctional probes attached to the cRLC, exploiting the fact that the native cRLCs in demembranated trabeculae can be efficiently replaced by labeled cRLCs with negligible effect on trabecular function (29, 30). We used in situ phosphorylation of cRLC by an expressed cMLCK to control the regulatory state of the thick filament and further exploited the cRLC exchange protocol to introduce cRLC mutants for mechanistic studies. We studied the relationship between thick and thin filament regulation using calcium titrations with probes on both the cRLC in the thick filaments and troponin in the thin filaments. The results presented below show that integration of thick and thin filament-based signaling pathways is essential for the normal regulation of contractility in the heart.     

Rational reprogramming of fungal polyketide first-ring cyclization

Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.Fungal polyketides are one of the largest families of structurally diverse natural products with antibiotic, antiproliferative, immunosuppressive, and enzyme inhibitory activities. Importantly, they also provide lead compounds and inspiration for pharmaceutical drug discovery, which is evidenced by the statin cholesterol-lowering agents (1, 2). Fungal polyketides are biosynthesized by multidomain megasynthases [type I iterative polyketide synthases (iPKSs)] that use ketoacyl synthase, acyl transferase, and acyl carrier protein (ACP) domains to catalyze recursive thio-Claisen condensations using malonyl-CoA extender units. Although the architecture of these enzymes is similar to a single module of the bacterial type I modular PKSs (3), fungal iPKSs use a single set of active sites iteratively, analogous to dissociated bacterial type II PKSs (4). Fungal iPKSs may be classified into three subgroups (5). Highly reducing iPKSs (hrPKSs) generate complex linear or nonaromatic cyclic products by reducing the nascent β-ketoacyl intermediates to the β-alcohol, the alkene, or the alkane after each condensation step using their ketoreductase, dehydratase, and enoyl reductase domains to execute a cryptic biosynthetic program (2, 6–8). Partially reducing iPKSs omit enoyl reduction to generate simple cyclic structures (5). Finally, nonreducing iPKSs (nrPKSs) feature no reducing domains and generate a wide variety of aromatic products. nrPKSs select different starter units by a starter unit:ACP transacylase domain (9) and mold the polyketide chains into cyclic products by regiospecific cyclizations. First-ring cyclizations are catalyzed by the product template domains (PTs) (10), whereas the polyketide chains are terminated by Claisen cyclase (11), macrolactone synthase (12) [thioesterase (TE)], or reductive release domains (2).Although the biosynthesis of most fungal polyketides requires a single iPKS enzyme, the assembly of the resorcylic acid lactones (RALs) involves a pair of collaborating hrPKSs and nrPKSs acting in sequence (12–17). Fungal RALs are rich pharmacophores with estrogen agonist (zearalenone), mitogen-activated protein kinase inhibitory (hypothemycin), and heat shock response modulatory activities [radicicol and monocillin II (1)] (Fig. 1) (18, 19). For these RALs, the hrPKS produces a reduced linear polyketide chain that is directly transferred to the nrPKS (9). The nrPKS further extends the polyketide, closes the first six-membered ring by aldol condensation, and releases the RAL product by macrolactone formation (Fig. 1). We have recently shown that the assembly of 10,11-dehydrocurvularin (2), a phytotoxic dihydroxyphenylacetic acid lactone (DAL) from Aspergillus terreus, uses a similar chemical modularity principle (20). Curvularins modulate the mammalian immune system by repressing the inducible nitric oxide synthase (21, 22). In addition, both monocillin II (1) and 10,11-dehydrocurvularin (2) act as promising broad spectrum inhibitors of various cancer cell lines in vitro by overwhelming the heat shock response, an evolutionarily conserved coping mechanism of eukaryotic cells that maintains protein homeostasis (23–26).Open in a separate windowFig. 1.Biosynthesis of monocillin II (1) and 10,11-dehydrocurvularin (2). (A) During the biosynthesis of the radicicol intermediate monocillin II (1) in C. chiversii (14), the hrPKS CcRADS1 produces a reduced pentaketide starter unit (13) that is transferred to the nrPKS CcRADS2 by the starter unit:ACP transacylase (SAT) domain (9) (step 1). After another four successive condensation events with malonyl-CoA (mCoA; step 2) catalyzed by the ketoacyl synthase of the nrPKS CcRADS2, the linear ACP-bound polyketide chain undergoes a C2-C7 aldol condensation catalyzed by the PT domain (10) (step 3). This condensation follows an F-type folding mode (28, 29); 1 is released by macrolactone formation catalyzed by the TE domain (2) (step 4). (B) Assembly of 2 in A. terreus AH-02–30-F7 also involves sequentially acting collaborating iPKSs. However, the hrPKS AtCURS1 produces a reduced tetraketide starter, whereas the AtCURS2 PT domain catalyzes aldol condensation in the C8-C3 register using an S-type folding mode (28). C-C bonds in bold indicate intact acetate equivalents (malonate-derived C₂ units) incorporated into the polyketide chain by the iPKSs.A crucial step during the programmed biosynthesis of aromatic polyketide natural products is the cyclization of the first ring, which is catalyzed by the PT domains of the nrPKS (10, 27). This event commits the highly reactive pluripotent poly-β-ketoacyl chains to defined structural classes of the possible polyketide scaffold space. PT-catalyzed cyclizations most often follow an F-type pattern, whereby the benzene ring is assembled from two intact malonate-derived C₂ units and two bridging carbons from two additional acetate equivalents (28, 29) (Fig. 1). F-type first-ring cyclizations typically result from aldol condensations in the C2-C7 (as in 1), C4-C9, or C6-C11 register. In contrast, bacterial polyketide cyclase/aromatase enzymes (parts of type II PKS multienzyme complexes) typically direct an S-type folding event, whereby the carbons of the benzene ring are derived from three intact malonate-derived C₂ units (1, 28). Nevertheless, a select few fungal polyketides, including DALs like 2, feature a first-ring connectivity that is analogous to the S-type folding mode, resulting from an unorthodox C8-C3 aldol cyclization event (20). The bacterial aromatase/cylcase enzymes show little sequence similarity to fungal PT domains and feature a different protein fold and active site architecture as a prominent example of convergent evolution (10, 30–32). The sequences of fungal PT domains catalyzing F-type cyclization can be classified into seven clades according to their regiospecificity and the length of their product (29, 33). Despite catalyzing an atypical S-type folding, the PT domain of the AtCURS2 curvularin synthase nrPKS is firmly rooted in the C2-C7 clade of PTs, which yield RALs like 1 (20). This result concurs with previous observations that fungal iPKSs evolve orthogonal product specificities primarily by point mutations and not domain shuffling among distinct enzymes (2).The present work aimed to define how different regiospecific outcomes for first-ring cyclization are programmed into nrPKS enzymes. By exploiting the orthogonal aldol condensation regiospecificities of the related PT domains of the nrPKSs for 1 and 2, we attempted to alter this program to switch F- and S-type cyclization modes. Achieving precise control of regiospecificity during the engineered biosynthesis of fungal polyketides is central to producing biologically active unnatural products, and it may guide efforts to generate novel chemical diversity from natural nonreduced fungal polyketides.     

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.     

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.     

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.     

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.     

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2′-deoxycytidine

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.Many viruses exhibit a high mutation rate when replicating their genomes, enabling quick adaptation to both changing cellular environments and therapeutics (1–5). Mammalian innate immune systems have developed a mechanism to exploit this high mutation rate against the virus; in a phenomenon termed “lethal mutagenesis,” (6–14) the immune system employs nucleic acid-modifying enzymes (e.g., APOBEC and ADAR) to increase the viral mutation rate sharply, stressing the functional gene product repertoire of the virus to the point that the viral population collapses (15–17). Several antiviral agents are proposed to work at least in part by a chemical version of lethal mutagenesis [e.g., ribavirin against hepatitis C virus (18–22), 5-hydroxy-2′-deoxycytidine against HIV (7), and T-705 against influenza viruses (23)]. When a sufficient number of these mutagenic nucleoside analogs is incorporated into viral genomes, the analogs increase the viral mutation rate above the error catastrophe limit, the rate above which no viable progeny are produced (6, 24–27). This work aimed to understand the molecular basis underlying the biological phenomenon of lethal mutagenesis induced by mutagenic nucleotides.The nucleoside analog 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212) (Fig. 1A) is specifically designed to induce lethal mutagenesis in HIV (28–30). KP1212, the only anti-HIV drug candidate in clinical trials to use this mechanism, has been shown to increase the mutation rate of HIV both in cell culture and in isolates from humans undergoing monotherapy (28, 29). The mutagenic properties of KP1212 in cell culture reveal that it is likely to base pair promiscuously with A and G, and that the progressive acquisition of mutations (primarily A-to-G and G-to-A transitions) precedes population collapse (Fig. 1B) (29). These data are supported by biochemical experiments performed using purified polymerases that establish the ability of KP1212 to pair with either A or G, both when the modified base enters DNA from the nucleotide pool and when it acts as a template base (30). Understanding the chemical and structural basis of mutagenesis of this drug candidate is critical for both its future clinical progress and the development of new therapeutic agents that work by the principle of lethal mutagenesis.Open in a separate windowFig. 1.Schematic presentation of KP1212''s mutagenic effect on viruses. (A) KP1212 exists as an array of different tautomeric forms, whereas cytosine almost exclusively exists as one form, the canonical keto-amino tautomer. (B) The deoxynucleotide analog of KP1212 is incorporated by viral polymerases, causing G-to-A and A-to-G mutations during viral replication. KP1212 is a poor substrate for human polymerases, which provides selectivity in its action against the virus. The progressive acquisition of mutations in the viral genome leads to viral population collapse.Tautomerism of KP1212 leading to viral mutagenesis has been proposed by us and others (29, 30) to be the basis for the clinical activity of this drug candidate. There are, however, no direct data to support that view. Tautomerism as the basis of mutagenesis of natural bases has long been proposed (31–35), and substantiated in part by experimental evidence of minor tautomeric forms of both canonical bases (36–38) and certain base analogs (e.g., 5-hydroxy-2′-deoxycytidine) (39). In a search for a chemical rationale to explain the ambiguous pairing of KP1212 during replication, the present study revealed that the compound readily adopts multiple tautomeric forms, some of which were unexpected. Previously, spectroscopic methods (e.g., UV, Raman, NMR) have been used to study tautomerism of nucleobases (37, 39, 40). In the current work, we also used a battery of spectroscopic tools (1D, 2D, and variable temperature NMR; FTIR; and 2D IR) (41) to quantify and structurally characterize the array of tautomers exhibited by KP1212. Tautomer interconversion equilibria deconvoluted from NMR spectra provided data on the relative levels of tautomers in solution. In parallel with the spectroscopic studies, the qualitative and quantitative features of KP1212 mutagenesis were directly determined by inserting the KP1212 base into a single-stranded viral vector and measuring the intrinsic mutagenic properties of the base, both in vitro and in vivo. Finally, a model is proposed that correlates the mutagenic and clinical properties of KP1212 with its ability to exist as multiple tautomers.