Role of Active Site Loop Dynamics in Mediating Ligand Release from E. coli Dihydrofolate Reductase.

Conformational fluctuations from ground-state to sparsely populated but functionally important excited states play a key role in enzyme catalysis. For Escherichia coli dihydrofolate reductase (DHFR), the release of the product tetrahydrofolate (THF) and oxidized cofactor NADP+ occurs through exchange between closed and occluded conformations of the Met20 loop. A "dynamic knockout" mutant of E. coli DHFR, where the E. coli sequence in the Met20 loop is replaced by the human sequence (N23PP/S148A), models human DHFR and is incapable of accessing the occluded conformation. 1H and 15N CPMG relaxation dispersion analysis for the ternary product complex of the mutant enzyme with NADP+ and the product analogue 5,10-dideazatetrahydrofolate (ddTHF) (E:ddTHF:NADP+) reveals the mechanism by which NADP+ is released when the Met20 loop cannot undergo the closed-to-occluded conformational transition. Two excited states were observed: one related to a faster, relatively high-amplitude conformational fluctuation in areas near the active site, associated with the shuttling of the nicotinamide ring of the cofactor out of the active site, and the other to a slower process where ddTHF undergoes small-amplitude motions within the binding site that are consistent with disorder observed in a room-temperature X-ray crystal structure of the N23PP/S148A mutant protein. These motions likely arise due to steric conflict of the pterin ring of ddTHF with the ribose-nicotinamide moiety of NADP+ in the closed active site. These studies demonstrate that site-specific kinetic information from relaxation dispersion experiments can provide intimate details of the changes in catalytic mechanism that result from small changes in local amino acid sequence.

Modeling of Hidden Structures Using Sparse Chemical Shift Data from NMR Relaxation Dispersion.

NMR relaxation dispersion measurements report on conformational changes occurring on the µs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a "sample and select" method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls (1H, 15N, and 13C'). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the µs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution.

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide-dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei.

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.

REVEL and BayesDel outperform other in silico meta-predictors for clinical variant classification.

Many in silico predictors of genetic variant pathogenicity have been previously developed, but there is currently no standard application of these algorithms for variant assessment. Using 4,094 ClinVar-curated missense variants in clinically actionable genes, we evaluated the accuracy and yield of benign and deleterious evidence in 5 in silico meta-predictors, as well as agreement of SIFT and PolyPhen2, and report the derived thresholds for the best performing predictor(s). REVEL and BayesDel outperformed all other meta-predictors (CADD, MetaSVM, Eigen), with higher positive predictive value, comparable negative predictive value, higher yield, and greater overall prediction performance. Agreement of SIFT and PolyPhen2 resulted in slightly higher yield but lower overall prediction performance than REVEL or BayesDel. Our results support the use of gene-level rather than generalized thresholds, when gene-level thresholds can be estimated. Our results also support the use of 2-sided thresholds, which allow for uncertainty, rather than a single, binary cut-point for assigning benign and deleterious evidence. The gene-level 2-sided thresholds we derived for REVEL or BayesDel can be used to assess in silico evidence for missense variants in accordance with current classification guidelines.

Utilizing dipole-dipole cross-correlated relaxation for the measurement of angles between pairs of opposing CαHα-CαHα bonds in anti-parallel ß-sheets.

Dipole-dipole cross-correlated relaxation (CCR) between two spin pairs is rich with macromolecular structural and dynamic information on inter-nuclear bond vectors. Measurement of short range dipolar CCR rates has been demonstrated for a variety of inter-nuclear vector spin pairs in proteins and nucleic acids, where the multiple quantum coherence necessary for observing the CCR rate is created by through-bond scalar coupling. In principle, CCR rates can be measured for any pair of inter-nuclear vectors where coherence can be generated between one spin of each spin pair, regardless of both the distance between the two spin pairs and the distance of the two spins forming the multiple quantum coherence. In practice, however, long range CCR (lrCCR) rates are challenging to measure due to difficulties in linking spatially distant spin pairs. By utilizing through-space relaxation allowed coherence transfer (RACT), we have developed a new method for the measurement of lrCCR rates involving CαHα bonds on opposing anti-parallel ß-strands. The resulting lrCCR rates are straightforward to interpret since only the angle between the two vectors modulates the strength of the interference effect. We applied our lrCCR measurement to the third immunoglobulin-binding domain of the streptococcal protein G (GB3) and utilize published NMR ensembles and static NMR/X-ray structures to highlight the relationship between the lrCCR rates and the CαHα-CαHα inter-bond angle and bond mobility. Furthermore, we employ the lrCCR rates to guide the selection of sub-ensembles from the published NMR ensembles for enhancing the structural and dynamic interpretation of the data. We foresee this methodology for measuring lrCCR rates as improving the generation of structural ensembles by providing highly accurate details concerning the orientation of CαHα bonds on opposing anti-parallel ß-strands.

Slow Dynamics of Tryptophan-Water Networks in Proteins.

Water has a profound effect on the dynamics of biomolecules and governs many biological processes, leading to the concept that function is slaved to solvent dynamics within and surrounding the biomolecule. Protein conformational changes on µs-ms time scales are frequently associated with protein function, but little is known about the behavior of protein-bound water on these time scales. Here we have used NMR relaxation dispersion measurements to probe the tryptophan indoles in the enzyme dihydrofolate reductase (DHFR). We find that during structural changes on the µs-ms time scale, large chemical shift changes are often observed for the NH proton on the indole ring, while relatively smaller chemical shift changes are observed for the ring nitrogen atom. Comparison with experimental chemical shifts and density functional theory-based chemical shift predictions show that during the structural change the tryptophan indole NHs remain bound to water, but the geometry of the protein-bound water networks changes. These results establish that relaxation dispersion measurements can indirectly probe water dynamics and indicate that water can influence, or be influenced by, protein conformational changes on the µs-ms time scale. Our data show that structurally conserved bound water molecules can play a critical role in transmitting information between functionally important regions of the protein and provide evidence that internal protein motions can be coupled through the mediation of hydrogen-bonded water bound in the protein structure.

Regulation of Androgen Receptor Activity by Transient Interactions of Its Transactivation Domain with General Transcription Regulators.

The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.

Detection of Correlated Protein Backbone and Side-Chain Angle Fluctuations.

NMR methods for the characterization of local protein motions have attained a high level of sophistication. Measurement of the synchronization between those motions, however, poses a serious challenge. Such correlated motions are one of the underlying mechanisms for the propagation of local changes to remote sites and as such for information transfer. Here, we demonstrate the experimental detection of the synchronization of motion over an intermediate range. To that purpose, we designed pulse sequences for the measurement of cross-correlated relaxation between the backbone HN -N and side-chain Hß -Cß dipoles in Ile, Thr, and Val in the protein GB3. These bonds are related through two and three intervening dihedral angles. We show that the correlated motions inherent in a structural ensemble obtained from a large and diverse array of NMR probes are in excellent agreement with our measurements.

Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion.

The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.

NMR reveals a dynamic allosteric pathway in thrombin.

Although serine proteases are found ubiquitously in both eukaryotes and prokaryotes, and they comprise the largest of all of the peptidase families, their dynamic motions remain obscure. The backbone dynamics of the coagulation serine protease, apo-thrombin (S195M-thrombin), were compared to the substrate-bound form (PPACK-thrombin). R1, R2, 15N-{1H}NOEs, and relaxation dispersion NMR experiments were measured to capture motions across the ps to ms timescale. The ps-ns motions were not significantly altered upon substrate binding. The relaxation dispersion data revealed that apo-thrombin is highly dynamic, with µs-ms motions throughout the molecule. The region around the N-terminus of the heavy chain, the Na+-binding loop, and the 170 s loop, all of which are implicated in allosteric coupling between effector binding sites and the active site, were dynamic primarily in the apo-form. Most of the loops surrounding the active site become more ordered upon PPACK-binding, but residues in the N-terminal part of the heavy chain, the γ-loop, and anion-binding exosite 1, the main allosteric binding site, retain µs-ms motions. These residues form a dynamic allosteric pathway connecting the active site to the main allosteric site that remains in the substrate-bound form.

EPI-001, A Compound Active against Castration-Resistant Prostate Cancer, Targets Transactivation Unit 5 of the Androgen Receptor.

Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.

Direct Investigation of Slow Correlated Dynamics in Proteins via Dipolar Interactions.

The synchronization of native state motions as they transition between microstates influences catalysis kinetics, mediates allosteric interactions, and reduces the conformational entropy of proteins. However, it has proven difficult to describe native microstates because they are usually minimally frustrated and may interconvert on the micro- to millisecond time scale. Direct observation of concerted equilibrium fluctuations would therefore be an important tool for describing protein native states. Here we propose a strategy that relates NMR cross-correlated relaxation (CCR) rates between dipolar interactions to residual dipolar couplings (RDCs) of individual consecutive H(N)-N and H(α)-C(α) bonds, which act as a proxy for the peptide planes and the side chains, respectively. Using Xplor-NIH ensemble structure calculations restrained with the RDC and CCR data, we observe collective motions on time scales slower than nanoseconds in the backbone for GB3. To directly access the correlations from CCR, we develop a structure-free data analysis. The resulting dynamic correlation map is consistent with the ensemble-restrained simulations and reveals a complex network. In general, we find that the bond motions are on average slightly correlated and that the local environment dominates many observations. Despite this, some patterns are typical over entire secondary structure elements. In the ß-sheet, nearly all bonds are weakly correlated, and there is an approximately binary alternation in correlation intensity corresponding to the solvent exposure/shielding alternation of the side chains. For α-helices, there is also a weak correlation in the H(N)-N bonds. The degree of correlation involving H(α)-C(α) bonds is directly affected by side-chain fluctuations, whereas loops show complex and nonuniform behavior.

Kinetics of the Antibody Recognition Site in the Third IgG-Binding Domain of Protein G.

Protein dynamics occurring on a wide range of timescales play a crucial role in governing protein function. Particularly, motions between the globular rotational correlation time (τc ) and 40 µs (supra-τc window), strongly influence molecular recognition. This supra-τc window was previously hidden, owing to a lack of experimental methods. Recently, we have developed a high-power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 µs. For the first time, this method, performed under super-cooled conditions, enabled us to detect a global motion in the first ß-turn of the third IgG-binding domain of protein G (GB3), which was extrapolated to 371±115 ns at 310 K. Furthermore, the same residues show the plasticity in the model-free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra-τc dynamics. This ß-turn is involved in antibody binding, exhibiting the potential link of the observed supra-τc motion with molecular recognition.

Classic Analysis of Biopolymer Dynamics Is Model Free.

Early analysis of biopolymer dynamics relied on a variety of motional models that were difficult to distinguish and sometimes gave contradictory results. The Lipari-Szabo model-free approach (documented in a 1980 article in Biophysical Journal, as well as in two more comprehensive 1982 articles in the Journal of the American Chemical Society, provided a simple formalism that allowed investigators to understand fluorescence and NMR experimental data without having to specify a motional model. Although the model-free method is not universally applicable (for example, its assumption of a uniform correlation time for overall molecular tumbling can be problematic for biomolecules containing areas of disorder), it remains the most popular and widely used technique for analyzing molecular motion.

Multi-probe relaxation dispersion measurements increase sensitivity to protein dynamics.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements are a valuable tool for the characterization of structural transitions on the micro-millisecond timescale. While the measurement of (15)N relaxation dispersion is now routine, the measurements with alternative nuclei remain limited. Here we report (15)N as well as (1)H R2 relaxation dispersion measurements of the N23PP/S148A "dynamic knockout" mutant of dihydrofolate reductase. The (1)H dispersion measurements are complementary to (15)N data as many additional residues are observed to have dispersive behavior for the (1)H nucleus. Simultaneous fitting of the dispersion profiles for the two nuclei increases the accuracy of exchange parameters determined for individual residues and clustered groups of residues. The different sensitivity of the two nuclei to changes in backbone torsional angles, ring currents, and hydrogen bonding effects provides important insights into the nature of the structural changes that take place during the exchange process. We observe clear evidence of direct and indirect hydrogen bond effects for the (15)N and (1)H chemical shift changes in the active-site, modulation of ring current shielding in the CD-loop and backbone torsional changes in a cluster of residues associated with the C-terminus. This work demonstrates the power of combined (1)H and (15)N probes for the study of backbone dynamics on the micro-millisecond timescale though the analysis of chemical shift changes.

Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway.

The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP(+)). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP(+) and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants.

Accurate scoring of non-uniform sampling schemes for quantitative NMR.

Non-uniform sampling (NUS) in NMR spectroscopy is a recognized and powerful tool to minimize acquisition time. Recent advances in reconstruction methodologies are paving the way for the use of NUS in quantitative applications, where accurate measurement of peak intensities is crucial. The presence or absence of NUS artifacts in reconstructed spectra ultimately determines the success of NUS in quantitative NMR. The quality of reconstructed spectra from NUS acquired data is dependent upon the quality of the sampling scheme. Here we demonstrate that the best performing sampling schemes make up a very small percentage of the total randomly generated schemes. A scoring method is found to accurately predict the quantitative similarity between reconstructed NUS spectra and those of fully sampled spectra. We present an easy-to-use protocol to batch generate and rank optimal Poisson-gap NUS schedules for use with 2D NMR with minimized noise and accurate signal reproduction, without the need for the creation of synthetic spectra.

Correlated motions are a fundamental property of ß-sheets.

Correlated motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. The mechanisms that underlie these processes remain largely unknown due mainly to limitations in their direct detection. Here, based on a detailed analysis of protein structures deposited in the protein data bank, as well as on state-of-the art molecular simulations, we provide general evidence for the transfer of structural information by correlated backbone motions, mediated by hydrogen bonds, across ß-sheets. We also show that the observed local and long-range correlated motions are mediated by the collective motions of ß-sheets and investigate their role in large-scale conformational changes. Correlated motions represent a fundamental property of ß-sheets that contributes to protein function.

Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR.

Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond-nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S(2)) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements.

Validated Conformational Ensembles Are Key for the Successful Prediction of Protein Complexes.

Conformational fluctuations in proteins play key roles in their functions and interactions. In this work, validated conformational ensembles for ubiquitin have been used in docking trials. The ensembles were used in a systematic predictive study of known ubiquitin complexes by applying a cross-docking strategy against the bound structure of each partner. The global docking predictions obtained with the complete ubiquitin ensembles were significantly better than those obtained with the crystallographic structure of free ubiquitin. Importantly, in all cases we identified an individual ensemble member that performed equally well, or even better, than the bound structure of ubiquitin. These results unequivocally demonstrate that, for proteins that recognize binding partners by conformational selection, the availability of conformational ensembles can greatly improve the performance of automatic docking predictions. Our results highlight the need for docking methodologies to capitalize on validated ensemble representations of biomacromolecules.