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Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å -1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å -1 , but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H 2 O and in D 2 O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å -1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set., (open access.)

KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, K d , for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. All rights reserved.)

We describe the conformational ensemble of the single-stranded r(UCAAUC) oligonucleotide obtained using extensive molecular dynamics (MD) simulations and Rosetta's FARFAR2 algorithm. The conformations observed in MD consist of A-form-like structures and variations thereof. These structures are not present in the pool generated using FARFAR2. By comparing with available nuclear magnetic resonance (NMR) measurements, we show that the presence of both A-form-like and other extended conformations is necessary to quantitatively explain experimental data. To further validate our results, we measure solution X-ray scattering (SAXS) data on the RNA hexamer and find that simulations result in more compact structures than observed from these experiments. The integration of simulations with NMR via a maximum entropy approach shows that small modifications to the MD ensemble lead to an improved description of the conformational ensemble. Nevertheless, we identify persisting discrepancies in matching experimental SAXS data., (© 2022 Bergonzo et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP., (© 2021. The Author(s).)

The respiratory syncytial virus (RSV) fusion (F) protein/polysorbate 80 (PS80) nanoparticle vaccine is the most clinically advanced vaccine for maternal immunization and protection of newborns against RSV infection. It is composed of a near-full-length RSV F glycoprotein, with an intact membrane domain, formulated into a stable nanoparticle with PS80 detergent. To understand the structural basis for the efficacy of the vaccine, a comprehensive study of its structure and hydrodynamic properties in solution was performed. Small-angle neutron scattering experiments indicate that the nanoparticle contains an average of 350 PS80 molecules, which form a cylindrical micellar core structure and five RSV F trimers that are arranged around the long axis of the PS80 core. All-atom models of full-length RSV F trimers were built from crystal structures of the soluble ectodomain and arranged around the long axis of the PS80 core, allowing for the generation of an ensemble of conformations that agree with small-angle neutron and X-ray scattering data as well as transmission electron microscopy (TEM) images. Furthermore, the hydrodynamic size of the RSV F nanoparticle was found to be modulated by the molar ratio of PS80 to protein, suggesting a mechanism for nanoparticle assembly involving addition of RSV F trimers to and growth along the long axis of the PS80 core. This study provides structural details of antigen presentation and conformation in the RSV F nanoparticle vaccine, helping to explain the induction of broad immunity and observed clinical efficacy. Small-angle scattering methods provide a general strategy to visualize surface glycoproteins from other pathogens and to structurally characterize nanoparticle vaccines.

Determination of structure of RNA via NMR is complicated in large part by the lack of a precise parameterization linking the observed chemical shifts to the underlying geometric parameters. In contrast to proteins, where numerous high-resolution crystal structures serve as coordinate templates for this mapping, such models are rarely available for smaller oligonucleotides accessible via NMR, or they exhibit crystal packing and counter-ion binding artifacts that prevent their use for the chemical shifts analysis. On the other hand, NMR-determined structures of RNA often are not solved at the density of restraints required to precisely define the variable degrees of freedom. In this study we sidestep the problems of direct parameterization of the RNA chemical shifts/structure relationship and examine the effects of imposing local fragmental coordinate similarity restraints based on similarities of the experimental secondary ribose 13 C/ 1 H chemical shifts instead. The effect of such chemical shift similarity (CSS) restraints on the structural accuracy is assessed via residual dipolar coupling (RDC)-based cross-validation. Improvements in the coordinate accuracy are observed for all of the six RNA constructs considered here as test cases, which argues for routine inclusion of these terms during NMR-based oligonucleotide structure determination. Such accuracy improvements are expected to facilitate derivation of the chemical shift/structure relationships for RNA., (© 2020 Lawrence and Grishaev; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric ( Sym CDTb; 3.14 Å) and an asymmetric form ( Asym CDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For Asym CDTb, a Ca 2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile ., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Molecular dynamics (MD) simulations play an important role in characterizing Ribonucleic Acid (RNA) structure, augmenting information from experimental techniques such as Nuclear Magnetic Resonance (NMR). In this work, we examine the accuracy of structural representation resulting from application of a number of explicit and implicit solvent models and refinement protocols against experimental data ranging from high density of residual dipolar coupling (RDC) restraints to completely unrestrained simulations. For a prototype A-form RNA helix, our results indicate that AMBER RNA force field with either implicit or explicit solvent can produce a realistic dynamic representation of RNA helical structure, accurately cross-validating with respect to a diverse array of NMR observables. In refinement against NMR distance restraints, modern MD force fields are found to be equally adequate, with high fidelity cross-validation to the residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), while slightly over-estimating structural order as monitored via NMR relaxation data. With restraints trimmed to encode only for base pairing information, cross-validation quality significantly deteriorates, now exhibiting a pronounced dependence on the choice of the solvent model. This deterioration is found to be partially reversible by increasing planarity restraints on the nucleobase geometry. For completely unrestrained MD simulations, the choice of water model becomes very important, with the best-performing TIP4P-Ew accurately reproducing both the RDC and RCSA data, while closely matching the NMR-derived order parameters. The information provided here will serve as a foundation for MD-based refinement of solution state NMR structures of RNA., (Published by Elsevier Inc.)

Structural information about ribonucleic acid (RNA) is lagging behind that of proteins, in part due to its high charge and conformational variability. Molecular dynamics (MD) has played an important role in describing RNA structure, complementing information from both nuclear magnetic resonance (NMR), or X-ray crystallography. We examine the impact of the choice of the empirical force field for RNA structure refinement using cross-validation against residual dipolar couplings (RDCs) as structural accuracy reporter. Four force fields, representing both the state-of-the art in RNA simulation and the most popular selections in NMR structure determination, are compared for a prototypical A-RNA helix. RNA structural accuracy is also evaluated as a function of both density and nature of input NMR data including RDCs, anisotropic chemical shifts, and distance restraints. Our results show a complex interplay between the experimental restraints and the force fields indicating two best-performing choices: high-fidelity refinement in explicit solvent, and the conformational database-derived potentials. Accuracy of RNA models closely tracks the density of 1-bond C-H RDCs, with other data types having beneficial, but smaller effects. At lower RDC density, or when refining against NOEs only, the two selected force fields are capable of accurately describing RNA helices with little or no experimental RDC data, making them available for the higher order structure assembly or better quantification of the intramolecular dynamics. Unrestrained simulations of simple RNA motifs with state-of-the art MD force fields appear to capture the flexibility inherent in nucleic acids while also maintaining a good agreement with the experimental observables.

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.

Riback et al (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Förster resonance energy transfer (FRET) experiments. There is thus no "contradiction" between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction., (Copyright © 2018, American Association for the Advancement of Science.)

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1's activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interactions drive transient formation of an N-terminal loop, the destabilization of which accounts for the dramatic change in size upon hyperphosphorylation. Phosphorylation also changes the preference of secondary structure formation of the PAGE4 ensemble, which leads to a transition between states that display different degrees of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions ofdifferent phosphoforms of PAGE4 with AP-1 and its downstream target, the androgen receptor (AR)-a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4, and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching of PAGE4 may potentially affect the efficiency of therapeutically targeting AR activity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg 2+ or Mn 2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn 2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1A cat , complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1A cat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1A cat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

Using fine-tuned hydrogen bonding criteria, a library of coiled peptide fragments has been generated from a large set of high-resolution protein X-ray structures. This library is shown to be an improved representation of ϕ/ψ torsion angles seen in intrinsically disordered proteins (IDPs). The ϕ/ψ torsion angle distribution of the library, on average, provides good agreement with experimentally observed chemical shifts and 3 J HN-Hα coupling constants for a set of five disordered proteins. Inspection of the coil library confirms that nearest-neighbor effects significantly impact the ϕ/ψ distribution of residues in the coil state. Importantly, 3 J HN-Hα coupling constants derived from the nearest-neighbor modulated backbone ϕ distribution in the coil library show improved agreement to experimental values, thereby providing a better way to predict 3 J HN-Hα coupling constants for IDPs, and for identifying locations that deviate from fully random behavior., (© 2017 The Protein Society. This article is a U.S. Government work and is in the public domain in the USA.)

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy therefore challenges our understanding of denaturation mechanism and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favorable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely, that the commonly used FRET chromophores do not qualitatively alter the results and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.

Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Accurate quantitative measurement of structural dispersion in proteins remains a prime challenge to both X-ray crystallography and NMR spectroscopy. Here we use a model-free approach based on measurement of many residual dipolar couplings (RDCs) in differentially orienting aqueous liquid crystalline solutions to obtain the side chain χ1 distribution sampled by each residue in solution. Applied to the small well-ordered model protein GB3, our approach reveals that the RDC data are compatible with a single narrow distribution of side chain χ1 angles for only about 40% of the residues. For more than half of the residues, populations greater than 10% for a second rotamer are observed, and four residues require sampling of three rotameric states to fit the RDC data. In virtually all cases, sampled χ1 values are found to center closely around ideal g(-), g(+) and t rotameric angles, even though no rotamer restraint is used when deriving the sampled angles. The root-mean-square difference between experimental (3)JHαHβ couplings and those predicted by the Haasnoot-parametrized, motion-adjusted Karplus equation reduces from 2.05 to 0.75 Hz when using the new rotamer analysis instead of the 1.1-Å X-ray structure as input for the dihedral angles. A comparison between observed and predicted (3)JHαHβ values suggests that the root-mean-square amplitude of χ1 angle fluctuations within a given rotamer well is ca. 20°. The quantitatively defined side chain rotamer equilibria obtained from our study set new benchmarks for evaluating improved molecular dynamics force fields, and also will enable further development of quantitative relations between side chain chemical shift and structure.

Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/β (EIN(α/β)) subdomain to the downstream protein partner bound to the EIN(α) subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN(α/β) subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. The latter likely represents an intermediate in the open-to-closed transition.

(3) JC'C' and (3) JHNHα couplings are related to the intervening backbone torsion angle ${\varphi }$ by standard Karplus equations. Although these couplings are known to be affected by parameters other than ${\varphi }$, including H-bonding, valence angles and residue type, experimental results and quantum calculations indicate that the impact of these latter parameters is typically very small. The solution NMR structure of protein GB3, newly refined by using extensive sets of residual dipolar couplings, yields 50-60 % better Karplus equation agreement between ${\varphi }$ angles and experimental (3) JC'C' and (3) JHNHα values than does the high-resolution X-ray structure. In intrinsically disordered proteins, (3) JC'C' and (3) JHNHα couplings can be measured at even higher accuracy, and the impact of factors other than the intervening torsion angle on (3) J will be smaller than in folded proteins, making these couplings exceptionally valuable reporters on the ensemble of ${\varphi }$ angles sampled by each residue., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (Kd = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63-66 and 74-76, which is distinct from the region known to enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. For the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site., (Published by Elsevier Inc.)

Three-bond (3)J(C'C') and (3)J(HNHα) couplings in peptides and proteins are functions of the intervening backbone torsion angle ϕ. In well-ordered regions, (3)J(HNHα) is tightly correlated with (3)J(C'C'), but the presence of large ϕ angle fluctuations differentially affects the two types of couplings. Assuming the ϕ angles follow a Gaussian distribution, the width of this distribution can be extracted from (3)J(C'C') and (3)J(HNHα), as demonstrated for the folded proteins ubiquitin and GB3. In intrinsically disordered proteins, slow transverse relaxation permits measurement of (3)J(C'C') and (3)J(HNH) couplings at very high precision, and impact of factors other than the intervening torsion angle on (3)J will be minimal, making these couplings exceptionally valuable structural reporters. Analysis of α-synuclein yields rather homogeneous widths of 69 ± 6° for the ϕ angle distributions and (3)J(C'C') values that agree well with those of a recent maximum entropy analysis of chemical shifts, J couplings, and (1)H-(1)H NOEs. Data are consistent with a modest (≤30%) population of the polyproline II region.

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone (1)H-(15)N, (15)N-(13)C', (1)H(α)-(13)C(α), and (13)C'-(13)C(α) one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with (1)H-(15)N and (1)H(α)-(13)C(α) RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in (13)C' chemical shift, (3)JHNHα values, and (13)C(α)-(13)C(β) RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.

The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains, have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144-231) and crystal (residues 146-231) structures by ∼30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and we obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely populated configurations of the N-terminal domain within the capsid protein dimer.

Quorum sensing (QS) is a cell-to-cell communication system responsible for a variety of bacterial phenotypes including virulence and biofilm formation. QS is mediated by small molecules, autoinducers (AIs), including AI-2 that is secreted by both Gram-positive and -negative microbes. LsrR is a key transcriptional regulator that governs the varied downstream processes by perceiving AI-2 signal, but its activation via autoinducer-binding remains poorly understood. Here, we provide detailed regulatory mechanism of LsrR from the crystal structures in complexes with the native signal (phospho-AI-2, D5P) and two quorum quenching antagonists (ribose-5-phosphate, R5P; phospho-isobutyl-AI-2, D8P). Interestingly, the bound D5P and D8P molecules are not the diketone forms but rather hydrated, and the hydrated moiety forms important H-bonds with the carboxylate of D243. The D5P-binding flipped out F124 of the binding pocket, and resulted in the disruption of the dimeric interface-1 by unfolding the α7 segment. However, the same movement of F124 by the D8P'-binding did not cause the unfolding of the α7 segment. Although the LsrR-binding affinity of R5P (Kd, ∼1 mM) is much lower than that of D5P and D8P (∼2.0 and ∼0.5 μM), the α-anomeric R5P molecule fits into the binding pocket without any structural perturbation, and thus stabilizes the LsrR tetramer. The binding of D5P, not D8P and R5P, disrupted the tetrameric structure and thus is able to activate LsrR. The detailed structural and mechanistic insights from this study could be useful for facilitating design of new antivirulence and antibiofilm agents based on LsrR.

In addition to the classic methods of structural biology--X-ray crystallography and NMR--solution X-ray scattering (SAXS) is playing an increasingly important role in structural investigation of biological macromolecules. However, the simultaneous ease of SAXS data collection and sophistication of its data analysis tools can present challenges to the investigator. Any sample, whether pure or contaminated, whether monodisperse or polydisperse, will yield scattering data, and it is up to the user to ensure the absence of artifacts and to choose a proper structural modeling strategy. This unit discusses experimental aspects of X-ray solution scattering, including sample preparation and data collection, as well as the steps in data processing and preliminary analysis required to ensure the absence of artifacts. The goal is to summarize everything than can go wrong with SAXS data measurement so the user can have confidence in the data before undertaking structural modeling., (© 2012 by John Wiley & Sons, Inc.)

The information content in 1-D solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique 3-D structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy-atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small- and wide-angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 Å. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca(2+) ions have been substituted by Pb(2+), and a target peptide. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data.

Hydrogen atom positions of nucleotide bases in RNA structures solved by X-ray crystallography are commonly derived from heavy-atom coordinates by assuming idealized geometries. In particular, N1-H1 vectors in G and N3-H3 vectors in U are commonly positioned to coincide with the bisectors of their respective heavy-atom angles. We demonstrate that quantum-mechanical optimization of the hydrogen positions relative to their heavy-atom frames considerably improves the fit of experimental residual dipolar couplings to structural coordinates. The calculations indicate that deviations of the imino N-H vectors in RNA U and G bases result from H-bonding within the base pair and are dominated by the attractive interaction between the H atom and the electron density surrounding the H-bond-acceptor atom. DFT optimization of H atom positions is impractical in structural biology studies. We therefore have developed an empirical relation that predicts imino N-H vector orientations from the heavy-atom coordinates of the base pair. This relation agrees very closely with the DFT results, permitting its routine application in structural studies.

The histological dye Congo Red (CR) previously has been shown to inhibit α-synuclein (aS) fibrillation, but the mode of this inhibition remained unclear. Because of favorable exchange kinetics, interaction between CR and aS lends itself to a detailed nuclear magnetic resonance study, and relaxation dispersion measurements yield the bound fraction and time scales for the interaction of aS with CR. We find that at pH 6, CR exists as a micelle, and at a CR:aS molar ratio of ~1, only a small fraction of aS (~2%) is bound to these micelles. Rapid exchange (k(ex) ~ 3000 s(-1)) between the free and CR-bound states broadens and strongly attenuates resonances of aS by two processes: a magnetic field-dependent contribution, caused by the chemical shift difference between the two states, and a nearly field-independent contribution caused by slower tumbling of aS bound to the CR micelle. The salt dependence of the interaction suggests a predominantly electrostatic mechanism for the 60 N-terminal residues, while the weaker interaction between residues 61-100 and CR is mostly hydrophobic. Chemical shift and transferred NOE data indicate that aS becomes slightly more helical but remains largely disordered when bound to CR. Results indicate that inhibition of fibril formation does not result from binding of CR to free aS and, therefore, must result from interaction of aS fibrils or protofibrils with CR micelles.

Analogous to the recently introduced ARTSY method for measurement of one-bond (1)H-(15)N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base (13)C-(1)H and (15)N-(1)H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in (1)H-(15)N and (13)C-(1)H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for (15)N-(1)H and 65/(S/N) Hz for (13)C-(1)H. The signal-to-noise ratio of both (1)H-(15)N and (1)H-(13)C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with (1)H-(1)H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.

The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN(α/β) subdomain and an aspartate (Asp129) on the EIN(α) subdomain results in a small (∼9°) reorientation of the EIN(α) and EIN(α/β) subdomains that is in turn propagated to a larger reorientation (∼26°) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

A new procedure, AXES, is introduced for fitting small-angle X-ray scattering (SAXS) data to macromolecular structures and ensembles of structures. By using explicit water models to account for the effect of solvent, and by restricting the adjustable fitting parameters to those that dominate experimental uncertainties, including sample/buffer rescaling, detector dark current, and, within a narrow range, hydration layer density, superior fits between experimental high resolution structures and SAXS data are obtained. AXES results are found to be more discriminating than standard Crysol fitting of SAXS data when evaluating poorly or incorrectly modeled protein structures. AXES results for ensembles of structures previously generated for ubiquitin show improved fits over fitting of the individual members of these ensembles, indicating these ensembles capture the dynamic behavior of proteins in solution.

The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS data that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C(2) symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large (∼70-90°) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.

Site-specific (1)H chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide moieties in the B3 domain of protein G (GB3). Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the (1)H(N) CSA tensor and either the (1)H-(15)N, the (1)H-(13)C', or the (1)H-(13)C(alpha) dipolar interactions. Analyses with the assumption that the (1)H(N) CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. (1)H(N) CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N-H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of N-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the (1)H(N) CSA. Using experimentally derived (1)H(N) CSA tensors, the optimal relaxation interference effect needed for narrowest (1)H(N) TROSY line widths is found at approximately 1200 MHz.

Site-specific (15)N chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide (15)N nuclei in the B3 domain of protein G (GB3) from residual chemical shift anisotropy (RCSA) measured in six different mutants that retain the native structure but align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension. This information is complemented by measurement of cross-correlated relaxation rates between the (15)N CSA tensor and either the (15)N-(1)H or (15)N-(13)C' dipolar interaction. In agreement with recent solid state NMR measurements, the (15)N CSA tensors exhibit only a moderate degree of variation from averaged values, but have larger magnitudes in alpha-helical (-173 +/- 7 ppm) than in beta-sheet (-162 +/- 6 ppm) residues, a finding also confirmed by quantum computations. The orientations of the least shielded tensor component cluster tightly around an in-peptide-plane vector that makes an angle of 19.6 +/- 2.5 degrees with the N-H bond, with the asymmetry of the (15)N CSA tensor being slightly smaller in alpha-helix (eta = 0.23 +/- 0.17) than in beta-sheet (eta = 0.31 +/- 0.11). The residue-specific (15)N CSA values are validated by improved agreement between computed and experimental (15)N R(1rho) relaxation rates measured for (15)N-{(2)H} sites in GB3, which are dominated by the CSA mechanism. Use of residue-specific (15)N CSA values also results in more uniform generalized order parameters, S(2), and predicts considerable residue-by-residue variations in the magnetic field strengths where TROSY line narrowing is most effective.

Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The ligand binding properties of the kringle 5 (K5) domain of human plasminogen have been investigated via intrinsic tryptophan fluorescence. The oleic acid (OA) affinity for K5 was quantified, yielding an association constant K(a) approximately 2.08 x 10(4) mM(-1). Simultaneously, it was determined that OA and trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) (K(a) approximately 50 mM(-1)) compete for binding to K5. The solution structure of K5 in the presence of 11 mM AMCHA was solved via NMR spectroscopy (protein heavy atom RMSD approximately 0.93 +/- 0.12 A). The AMCHA binding site was localized via (1)H/(15)N chemical shift perturbation mapping assisted by in silico docking. We have found that AMCHA binds at the canonical kringle lysine binding site (LBS), structured by the Pro54-Gly60 segment plus the neighboring Phe36, Thr37, Trp62, Leu71, and Tyr72 residues. The segment 30-42, encompassing LBS residues, appears to be endowed with a higher degree of structural flexibility as suggested by the relatively lower value of S(2), the generalized order parameter, consistent with a higher backbone heavy atom RMSD of approximately 1.22 A (vs 0.84 A overall) between the two monomeric units in the crystal unit cell, of potential significance for ligand binding. OA was found to perturb the same area of the protein, namely, the LBS, as well as Tyr74. Combined with previous studies, the observation of OA binding expands the range of ligands that interact with kringle 5 while it widens the scope of potential biological functions for kringle domains.

This paper describes an approach for making use of the components of the experimentally determined rotational diffusion tensor derived from NMR relaxation measurements in macromolecular structure determination. The parameters of the rotational diffusion tensor describe the shape and size of the macromolecule or macromolecular complex, and are therefore complementary to traditional NMR restraints. The structural information contained in the rotational diffusion tensor is not dissimilar to that present in the small-angle region of solution X-ray scattering profiles. We demonstrate the utility of rotational diffusion tensor restraints for protein structure refinement using the N-terminal domain of enzyme I (EIN) as an example and validate the results by solution small-angle X-ray scattering. We also show how rotational diffusion tensor restraints can be used for docking complexes using the dimeric HIV-1 protease and the EIN-HPr complexes as examples. In the former case, the rotational diffusion tensor restraints are sufficient in their own right to determine the position of one subunit relative to another. In the latter case, rotational diffusion tensor restraints complemented by highly ambiguous distance restraints derived from chemical shift perturbation mapping and a hydrophobic contact potential are sufficient to correctly dock EIN to HPr. In each case, the cluster containing the lowest-energy structure corresponds to the correct solution.

Knowledge of (15)N chemical shift anisotropy is prerequisite both for quantitative interpretation of nuclear spin relaxation rates in terms of local dynamics and for the use of residual chemical shift anisotropy (RCSA) as a constraint in structure determination. Accurate measurement of the very small RCSA from the difference in (15)N chemical shift under isotropic and weakly aligning liquid crystalline conditions is very sensitive to minute differences in sample conditions, such as pH or ionic strength. For this reason, chemical shifts were measured for the same solution, under static liquid crystalline alignment, and under magic angle spinning conditions where alignment relative to the magnetic field is removed. Measurements were made for 14 well-resolved G-N(1) and 6 U-N(3) (15)N nuclei in a sample of tRNA(Val). Fitting these RCSA data together with (15)N-(1)H dipole-CSA cross-correlated relaxation measurements to the recently refined structural model of tRNA(Val) yields the magnitude, asymmetry, and orientation of the (15)N CSA tensors.

Neurotrypsin is a multidomain protein that serves as a brain-specific serine protease. Here we report the NMR structure of its kringle domain, NT/K. The data analysis was performed with the BACUS (Bayesian analysis of coupled unassigned spins) algorithm. This study presents the first application of BACUS to the structure determination of a 13C unenriched protein for which no prior experimental 3D structure was available. NT/K adopts the kringle fold, consisting of an antiparallel beta-sheet bridged by an overlapping pair of disulfides. The structure reveals the presence of a surface-exposed left-handed polyproline II helix that is closely packed to the core beta-structure. This feature distinguishes NT/K from other members of the kringle fold and points toward a novel functional role for a kringle domain. Functional divergence among kringle domains is discussed on the basis of their surface and electrostatic characteristics.

A procedure is presented for refinement of a homology model of E. coli tRNA(Val), originally based on the X-ray structure of yeast tRNA(Phe), using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-H(N) RDCs measured with Pf1 phage alignment, and 20 imino N-H(N) RDCs obtained from magnetic field dependent alignment of tRNA(Val). The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA(Phe) by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q (free) = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA(Phe), in agreement with previous NMR-based tRNA(Val) models.

Neuroligins are post-synaptic cell adhesion molecules that promote synaptic maturation and stabilization upon binding with pre-synaptic partners, the alpha- and beta-neurexins. Using a combination of analytical ultracentrifugation, small angle X-ray, and neutron scattering, we have characterized the low-resolution three-dimensional structure of the extracellular domain of the neuroligins, free in solution, and in complex with beta-neurexin. The globular extracellular domain of the neuroligins forms stable homodimers through a four-helix bundle typical of the cholinesterases and other members of the alpha/beta-hydrolase fold family. The presence of the stalk region adds to the extracellular domain of neuroligin-1 an elongated structure, suggesting a rod-like nature of the stalk domain. Sedimentation equilibrium coupled with solution scattering data of the beta-neurexin/neuroligin-1 complex indicated a 2:2 stoichiometry where two beta-neurexin molecules bind to a neuroligin-1 dimer. Deuteration of neurexin allowed us to collect neutron scattering data that, in combination with other biochemical techniques, provide a basis for optimizing the positioning of each component in a detailed computational model of the neuroligin/neurexin complex. As several mutations of both neurexin and neuroligin genes have been linked to autism spectrum disorders and mental retardation, these new structures provide an important framework for the study of altered structure and function of these synaptic proteins.

TolR is a part of the Pal/Tol system which forms a five-member, membrane-spanning, multiprotein complex that is conserved in Gram-negative bacteria. The Pal/Tol system helps to maintain the integrity of the outer membrane and has been proposed to be involved in several other cellular processes including cell division. Obtaining the structure of TolR is of interest not only to help explain the many proposed functions of the Pal/Tol system but also to gain an understanding of the TolR homologues ExbD and MotB and to provide more targets for antibacterial treatments. In addition, the structure may provide insights into how colicins and bacteriophages are able to enter the cell. Here we report the solution structure of the homodimeric periplasmic domain of TolR from Haemophilus influenzae, determined with conventional, NOE-based NMR spectroscopy, supplemented by extensive residual dipolar coupling measurements. A novel method for assembling the dimer from small-angle X-ray scattering data confirms the NMR-derived structure. To facilitate NMR spectral analysis, a TolR construct containing residues 59-130 of the 139-residue protein was created. The periplasmic domain of TolR forms a C 2-symmetric dimer consisting of a strongly curved eight-stranded beta-sheet, generating a large deep groove on one side, while four helices cover the other face of the sheet. The structure of the TolR dimer together with data from the literature suggests how the periplasmic domain of TolR is most likely oriented relative to the cytoplasmic membrane and how it may interact with other components of the Pal/Tol system, particularly TolQ.

Determination of the accurate three-dimensional structure of large proteins by NMR remains challenging due to a loss in the density of experimental restraints resulting from the often prerequisite perdeuteration. Solution small-angle scattering, which carries long-range translational information, presents an opportunity to enhance the structural accuracy of derived models when used in combination with global orientational NMR restraints such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). We have quantified the improvements in accuracy that can be obtained using this strategy for the 82 kDa enzyme Malate Synthase G (MSG), currently the largest single chain protein solved by solution NMR. Joint refinement against NMR and scattering data leads to an improvement in structural accuracy as evidenced by a decrease from approximately 4.5 to approximately 3.3 A of the backbone rmsd between the derived model and the high-resolution X-ray structure, PDB code 1D8C. This improvement results primarily from medium-angle scattering data, which encode the overall molecular shape, rather than the lowest angle data that principally determine the radius of gyration and the maximum particle dimension. The effect of the higher angle data, which are dominated by internal density fluctuations, while beneficial, is also found to be relatively small. Our results demonstrate that joint NMR/SAXS refinement can yield significantly improved accuracy in solution structure determination and will be especially well suited for the study of systems with limited NMR restraints such as large proteins, oligonucleotides, or their complexes.

For base-paired nucleic acids, variations in (1) J (NH) and the imino (1)H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the (1) J (NH) coupling for the imino proton then can be approximated by (1) J (NH) = (1.21Hz/ppm)delta(H) - 103.5 +/- 0.6 Hz, where delta(H) represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of ((1) J (NH) + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA(Val) and the alignment tensor determined from magnetic-field alignment of tRNA(Val) agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases.