Direct use of unassigned resonances in NMR structure calculations with proxy residues

We present a method that significantly enhances the robustness of (automated) NMR structure determination by allowing the NOE data corresponding to unassigned NMR resonances to be used directly in the calculations. The unassigned resonances are represented by additional atoms or groups of atoms that have no interaction with the regular protein atoms except through distance restraints. These so-called "proxy" residues can be used to generate NOE-based distance restraints in a similar fashion as for the assigned part of the protein. If sufficient NOE information is available, the restraints are expected to place the proxies at positions close to the correct atoms for the unassigned resonance, which can facilitate subsequent assignment. Convergence can be further improved by supplying additional information about the possible identities of the unassigned resonances. We have implemented this approach in the widely used automated assignment and structure calculation protocols ARIA and CANDID. We find that it significantly increases the robustness of structure calculations with regard to missing assignments and yields structures of higher quality. Our approach is still able to find correctly folded structures with up to 30% randomly missing resonance assignments, and even when only backbone and beta resonances are present! This should be of significant value to NMR-based structural proteomics initiatives.     

Deuterated protein folds obtained directly from unassigned nuclear overhauser effect data

We demonstrate the feasibility of determining the global fold of a highly deuterated protein from unassigned experimental NMR nuclear Overhauser effect (NOE) data only. The method relies on the calculation of a spatial configuration of covalently unconnected protons-a "cloud"-directly from unassigned distance restraints derived from 13C- and 15N-edited NOESY spectra. Each proton in the cloud, labeled by its chemical shift and that of the directly bound 13C or 15N, is subsequently mapped to specific atoms in the protein. This is achieved via graph-theoretical protocols that search for connectivities in graphs that encode the structural information within the cloud. The peptidyl HN chain is traced by seeking for all possible routes and selecting the one that yields the minimal sum of sequential distances. Complete proton identification in the cloud is achieved by linking the side-chain protons to proximal main-chain HNs via bipartite graph matching. The identified protons automatically yield the NOE assignments, which in turn are used for structure calculation with RosettaNMR, a protocol that incorporates structural bias derived from protein databases. The method, named Sparse-Constraint CLOUDS, was applied to experimental NOESY data on the 58-residue Z domain of staphylococcal protein A. The generated structures are of similar accuracy to those previously reported, which were derived via a conventional approach involving a larger NMR data set. Additional tests were performed on seven reported protein structures of various folds, using restraint lists simulated from the known atomic coordinates.     

Stereospecific assignments of protein NMR resonances based on the tertiary structure and 2D/3D NOE data

In many cases of protein structure determination by NMR a high-quality structure is required. An important contribution to structural precision is stereospecific assignment of magnetically nonequivalent prochiral methylene and methyl groups, eliminating the need for introducing pseudoatoms and pseudoatom corrections in distance restraint lists. Here, we introduce the stereospecific assignment program that uses the resonance assignment, a preliminary 3D structure and 2D and/or 3D nuclear Overhauser effect spectroscopy peak lists for stereospecific assignment. For each prochiral group the algorithm automatically calculates a score for the two different stereospecific assignment possibilities, taking into account the presence and intensity of the nuclear Overhauser effect (NOE) peaks that are expected from the local environment of each prochiral group (i.e., the close neighbors). The performance of the algorithm has been tested and used on NMR data of alpha-helical and beta-sheet proteins using homology models and/or X-ray structures. The program produced no erroneous stereospecific assignments provided the NOEs were carefully picked and the 3D model was sufficiently accurate. The set of NOE distance restraints produced by nmr2st using the results of the SSA module was superior in generating good-quality ensembles of NMR structures (low deviations from upper limits in conjunction with low root-mean-square-deviation values) in the first round of structure calculations. The program uses a novel approach that employs the entire 3D structure of the protein to obtain stereospecific assignment; it can be used to speed up the NMR structure refinement and to increase the quality of the final NMR ensemble even when no scalar or residual dipolar coupling information is available.     

Conformation and absolute configuration of nocathiacin I determined by NMR spectroscopy and chiral capillary electrophoresis

Nocathiacin I (BMS-249524) is a highly cross-linked thiazolyl peptide that displays potent activity against Gram-positive bacteria, including a number of antibiotic-resistant strains. This natural product contains 10 chiral centers. NMR studies have been performed to characterize the solution structure of nocathiacin I. A uniformly 13C,15N-labeled sample was used to obtain NMR assignments. Restrained simulated annealing calculations were performed by using accurately determined NOE distance restraints. All of the chiral centers were allowed to float during the simulated annealing protocol. Two clusters of structures were obtained that satisfy the NOE restraints very well and that are reasonably consistent with vicinal J-coupling constants. Within each cluster, all 10 chiral centers are uniquely defined. The two clusters are effectively mirror images of each other: all chiral centers that have the R(S) configuration in one cluster have the S(R) configuration in the other. The single threonine residue in nocathiacin I was subsequently determined to be l-threonine by chiral capillary electrophoresis, allowing the absolute configurations of all 10 chiral centers to be defined.     

Impact of 15N R2/R1 relaxation restraints on molecular size, shape, and bond vector orientation for NMR protein structure determination with sparse distance restraints

(15)N R(2)/R(1) relaxation data contain information on molecular shape and size as well as on bond vector orientations relative to the diffusion tensor. Since the diffusion tensor can be directly calculated from the molecular coordinates, direct inclusion of (15)N R(2)/R(1) restraints in NMR structure calculations without any a priori assumptions is possible. Here we show that (15)N R(2)/R(1) restraints are particularly valuable when only sparse distance restraints are available. Using three examples of proteins of varying size, namely, GB3 (56 residues), ubiquitin (76 residues), and the N-terminal domain of enzyme I (EIN, 249 residues), we show that incorporation of (15)N R(2)/R(1) restraints results in large and significant increases in coordinate accuracy that can make the difference between being able or unable to determine an approximate global fold. For GB3 and ubiquitin, good coordinate accuracy was obtained using only backbone hydrogen-bond restraints supplemented by (15)N R(2)/R(1) relaxation restraints. For EIN, the global fold could be determined using sparse nuclear Overhauser enhancement (NOE) distance restraints involving only NH and methyl groups in conjunction with (15)N R(2)/R(1) restraints. These results are of practical significance in the study of larger and more complex systems, where the increasing spectral complexity and number of chemical shift degeneracies reduce the number of unambiguous NOE assignments that can be readily obtained, resulting in progressively reduced NOE coverage as the size of the protein increases.     

Automated protein structure determination from NMR spectra

Fully automated structure determination of proteins in solution (FLYA) yields, without human intervention, three-dimensional protein structures starting from a set of multidimensional NMR spectra. Integrating existing and new software, automated peak picking over all spectra is followed by peak list filtering, the generation of an ensemble of initial chemical shift assignments, the determination of consensus chemical shift assignments for all (1)H, (13)C, and (15)N nuclei, the assignment of NOESY cross-peaks, the generation of distance restraints, and the calculation of the three-dimensional structure by torsion angle dynamics. The resulting, preliminary structure serves as additional input to the second stage of the procedure, in which a new ensemble of chemical shift assignments and a refined structure are calculated. The three-dimensional structures of three 12-16 kDa proteins computed with the FLYA algorithm coincided closely with the conventionally determined structures. Deviations were below 0.95 A for the backbone atom positions, excluding the flexible chain termini. 96-97% of all backbone and side-chain chemical shifts in the structured regions were assigned to the correct residues. The purely computational FLYA method is suitable for substituting all manual spectra analysis and thus overcomes a main efficiency limitation of the NMR method for protein structure determination.     

Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments

Given the three-dimensional (3D) structure of a protein, the binding pose of a ligand can be determined using distance restraints derived from assigned intra-ligand and protein-ligand nuclear Overhauser effects (NOEs). A primary limitation of this approach is the need for resonance assignments of the ligand-bound protein. We have developed an approach that utilizes data from 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectra for evaluating ligand binding poses without requiring protein NMR resonance assignments. Only the 1H NMR assignments of the bound ligand are essential. Trial ligand binding poses are generated by any suitable method (e.g., computational docking). For each trial binding pose, the 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum is predicted, and the predicted and observed patterns of protein-ligand NOEs are matched and scored using a fast, deterministic bipartite graph matching algorithm. The best scoring (lowest "cost") poses are identified. Our method can incorporate any explicit restraints or protein assignment data that are available, and many extensions of the basic procedure are feasible. Only a single sample is required, and the method can be applied to both slowly and rapidly exchanging ligands. The method was applied to three test cases: one complex involving muscle fatty acid-binding protein (mFABP) and two complexes involving the leukocyte function-associated antigen 1 (LFA-1) I-domain. Without using experimental protein NMR assignments, the method identified the known binding poses with good accuracy. The addition of experimental protein NMR assignments improves the results. Our "NOE matching" approach is expected to be widely applicable; i.e., it does not appear to depend on a fortuitous distribution of binding pocket residues.     

Semiautomatic sequence-specific assignment of proteins based on the tertiary structure--the program st2nmr

The sequence-specific assignment of resonances is still the most time-consuming procedure that is necessary as the first step in high-resolution NMR studies of proteins. In many cases a reliable three-dimensional (3D) structure of the protein is available, for example, from X-ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence-specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance-dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an alpha-helical (cytochrome c) and beta-sheet (lipocalin) protein using homology models and/or X-ray structures; it succeeded in completely reproducing the correct sequence-specific assignments in most cases using 2D and/or 15N/13C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.     

The atom assignment problem in automated de novo drug design. 3. Algorithms for optimization of fragment placement onto 3D molecular graphs

Summary Atom assignment onto 3D molecular graphs is a combinatoric problem in discrete space. If atoms are to be placed efficiently on molecular graphs produced in drug binding sites, the assignment must be optimized. An algorithm, based on simulated annealing, is presented for efficient optimization of fragment placement. Extensive tests of the method have been performed on five ligands taken from the Protein Data Bank. The algorithm is presented with the ligand graph and the electrostatic potential as input. Self placement of molecular fragments was monitored as an objective test. A hydrogen-bond option was also included, to enable the user to highlight specific needs. The algorithm performed well in the optimization, with successful replications. In some cases, a modification was necessary to reduce the tendency to give multiple halogenated structures. This optimization procedure should prove useful for automated de novo drug design.     

Pepto: An expert system for automatic peak assignment of two-dimensional nuclear magnetic resonance spectra of proteins

Two-dimensional Nuclear Magnetic Resonance (2DFTNMR) is presently the most powerful tool to determine protein structures in solution. Peak assignment (an interpretation of the two-dimensional spectra that leads to the individuation of pairs of Hydrogen atoms that are involved in an NOE peak) is a cornerstone of such use of 2DFTNMR. Manual peak assignment of a protein often requires months of work by a specialized equipe. An automation of this task could speed up the protein study process, or alternatively allow to study previously unmanageable proteins. This article describes PEPTO, an expert system for the interpretation of sets of 2DFTNMR spectra on proteins. The present version of the program deals with spectra obtained from NOESY and COSY experiments. Tests of PEPTO on simulated spectra of five proteins with known assignments are also described and dicussed.     

Computational design of a single amino acid sequence that can switch between two distinct protein folds

The functions of many proteins are mediated by specific conformational changes, and therefore the ability to design primary sequences capable of secondary and tertiary changes is an important step toward the creation of novel functional proteins. To this end, we have developed an algorithm that can optimize a single amino acid sequence for multiple target structures. The algorithm consists of an outer loop, in which sequence space is sampled by a Monte Carlo search with simulated annealing, and an inner loop, in which the effect of a given mutation is evaluated on the various target structures by using the rotamer packing routine and composite energy function of the protein design software, RosettaDesign. We have experimentally tested the method by designing a peptide, Sw2, which can be switched from a 2Cys-2His zinc finger-like fold to a trimeric coiled-coil fold, depending upon the pH or the presence of transition metals. Physical characterization of Sw2 confirms that it is able to reversibly adopt each intended target fold.     

Solution structure of a peptide nucleic acid duplex from NMR data: features and limitations

This paper describes the results of a 1D and 2D NMR spectroscopy study of a palindromic 8-base pair PNA duplex GGCATGCC in H2O and H2O-D2O solutions. The (1)H NMR peaks have been assigned for most of the protons of the six central base pairs, as well as for several amide protons of the backbone. The resulting 36 interbase and base-backbone distance restraints were used together with Watson-Crick restraints to generate the PNA duplex structure in the course of 10 independent simulated annealing runs followed by restrained molecular dynamics (MD) simulations in explicit water. The resulting PNA structures correspond to a P-type helix with helical parameters close to those observed in the crystal structures of PNA. Based on the current limited number of restraints obtained from NMR spectra, alternative structures obtained by MD from starting PNA models based on DNA cannot be ruled out and are also discussed.     

Structures of protein-protein complexes are docked using only NMR restraints from residual dipolar coupling and chemical shift perturbations

NMR structures of protein-protein and protein-ligand complexes rely heavily on intermolecular NOEs. Recent work has shown that if no significant conformational changes occur upon complex formation residual dipolar coupling can replace most of the NOE restraints in protein-protein complexes, while restraints derived from chemical shift perturbations can largely replace intermolecular NOEs in protein-ligand structures. By combining restraints from chemical shift perturbations with orientation restraints derived from measurements of residual dipolar couplings, we show that the structure of the EIN-HPr complex can be calculated without NOE restraints. The final structure, built from the crystal structures of EIN and HPr in their uncomplexed form and docked only with NMR restraints, places HPr within 2.5 A of the position determined from the mean NMR structure of the complex.     

3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints

In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.     

Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics

A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure. The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations. The target function also comprises three nonbonded interactions terms: a van der Waals repulsion term to prevent atomic overlap, a radius of gyration term (E(rgyr)) to avoid expansion at the protein-protein interface, and a torsion angle database potential of mean force to bias interfacial side chain conformations toward physically allowed rotamers. For the EIN-HPr and IIA(Glc)-HPr complexes, all structures satisfying the experimental restraints (i.e., both the ambiguous intermolecular distance restraints and the dipolar couplings) converge to a single cluster with mean backbone coordinate accuracies of 0.7-1.5 A. For the IIA(Mtl)-HPr complex, twofold degeneracy remains, and the structures cluster into two distinct solutions differing by a 180 degrees rotation about the z axis of the alignment tensor. The correct and incorrect solutions which have mean backbone coordinate accuracies of approximately 0.5 and approximately 10.5 A, respectively, can readily be distinguished using a variety of criteria: (a) examination of the overall (1)H(N)/(15)N chemical shift perturbation map (because the incorrect cluster predicts the presence of residues at the interface that experience only minimal chemical shift perturbations; this information is readily incorporated into the calculations in the form of ambiguous intermolecular repulsion restraints); (b) back-calculation of dipolar couplings on the basis of molecular shape; or (c) the E(rgyr) distribution which, because of its global nature, directly reflects the interfacial packing quality. This methodology should be particularly useful for high throughput, NMR-based, structural proteomics.     

Computer-aided determination of relative stereochemistry and 3D models of complex organic molecules from 2D NMR spectra

A method for elucidation of the relative stereoconfiguration of natural product molecular structures and their 3D models based on NOE data and the application of a genetic algorithm is described. The method is applicable mainly for rigid polycyclic structures commonly encountered in natural products. It is demonstrated that the technique of simulated annealing cannot be easily used when dealing with low-weight fused ring molecules but the application of a genetic algorithm is proven successful. Examples of a typical genetic algorithm workflow and the optimization of the algorithmic parameters are discussed. The efficiency of the approach developed here is demonstrated on the complex natural products of both Taxol^® (C₄₇H₅₁NO₁₄) and brevetoxin B (C₅₀H₇₀O₁₄).     

PROSIGN: a method for protein secondary structure assignment based on three-dimensional coordinates of consecutive C(alpha) atoms

The automatic assignment of secondary structure from three-dimensional atomic coordinates of proteins is an essential step for the analysis and modeling of protein structures. So different methods based on different criteria have been designed to perform this task. We introduce a new method for protein secondary structure assignment based solely on C(alpha) coordinates. We introduce four certain relations between C(alpha) three-dimensional coordinates of consecutive residues, each of which applies to one of the four regular secondary structure categories: alpha-helix, 3(10)-helix, pi-helix and beta-strand. In our approach, the deviation of the C(alpha) coordinates of each residue from each relation is calculated. Based on these deviation values, secondary structures are assigned to all residues of a protein. We show that our method agrees well with popular methods as DSSP, STRIDE and assignments in PDB files. It is shown that our method gives more information about helix geometry leading to more accurate secondary structure assignment.     

CABS‐NMR—De novo tool for rapid global fold determination from chemical shifts,residual dipolar couplings and sparse methyl‐methyl noes

Recent development of nuclear magnetic resonance (NMR) techniques provided new types of structural restraints that can be successfully used in fast and low‐cost global protein fold determination. Here, we present CABS‐NMR, an efficient protein modeling tool, which takes advantage of such structural restraints. The restraints are converted from original NMR data to fit the coarse grained protein representation of the C‐Alpha‐Beta‐Side‐group (CABS) algorithm. CABS is a Monte Carlo search algorithm that uses a knowledge‐based force field. Its versatile structure enables a variety of protein‐modeling protocols, including purely de novo folding, folding guided by restraints derived from template structures or, structure assembly based on experimental data. In particular, CABS‐NMR uses the distance and angular restraints set derived from various NMR experiments. This new modeling technique was successfully tested in structure determination of 10 globular proteins of size up to 216 residues, for which sparse NMR data were available. Additional detailed analysis was performed for a S100A1 protein. Namely, we successfully predicted Nuclear Overhauser Effect signals on the basis of low‐energy structures obtained from chemical shifts by CABS‐NMR. It has been observed that utility of chemical shifts and other types of experimental data (i.e. residual dipolar couplings and methyl‐methyl Nuclear Overhauser Effect signals) in the presented modeling pipeline depends mainly on size of a protein and complexity of its topology. In this work, we have provided tools for either post‐experiment processing of various kinds of NMR data or fast and low‐cost structural analysis in the still challenging field of new fold predictions. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Refinement of NMR structures using implicit solvent and advanced sampling techniques

NMR biomolecular structure calculations exploit simulated annealing methods for conformational sampling and require a relatively high level of redundancy in the experimental restraints to determine quality three-dimensional structures. Recent advances in generalized Born (GB) implicit solvent models should make it possible to combine information from both experimental measurements and accurate empirical force fields to improve the quality of NMR-derived structures. In this paper, we study the influence of implicit solvent on the refinement of protein NMR structures and identify an optimal protocol of utilizing these improved force fields. To do so, we carry out structure refinement experiments for model proteins with published NMR structures using full NMR restraints and subsets of them. We also investigate the application of advanced sampling techniques to NMR structure refinement. Similar to the observations of Xia et al. (J.Biomol. NMR 2002, 22, 317-331), we find that the impact of implicit solvent is rather small when there is a sufficient number of experimental restraints (such as in the final stage of NMR structure determination), whether implicit solvent is used throughout the calculation or only in the final refinement step. The application of advanced sampling techniques also seems to have minimal impact in this case. However, when the experimental data are limited, we demonstrate that refinement with implicit solvent can substantially improve the quality of the structures. In particular, when combined with an advanced sampling technique, the replica exchange (REX) method, near-native structures can be rapidly moved toward the native basin. The REX method provides both enhanced sampling and automatic selection of the most native-like (lowest energy) structures. An optimal protocol based on our studies first generates an ensemble of initial structures that maximally satisfy the available experimental data with conventional NMR software using a simplified force field and then refines these structures with implicit solvent using the REX method. We systematically examine the reliability and efficacy of this protocol using four proteins of various sizes ranging from the 56-residue B1 domain of Streptococcal protein G to the 370-residue Maltose-binding protein. Significant improvement in the structures was observed in all cases when refinement was based on low-redundancy restraint data. The proposed protocol is anticipated to be particularly useful in early stages of NMR structure determination where a reliable estimate of the native fold from limited data can significantly expedite the overall process. This refinement procedure is also expected to be useful when redundant experimental data are not readily available, such as for large multidomain biomolecules and in solid-state NMR structure determination.