Xplor-NIH: Better parameters and protocols for NMR protein structure determination.

The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor-NIH biomolecular structure determination package. In combination with an improved treatment of selected non-bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters are tested using structure calculations on 30 proteins with restraints derived from nuclear magnetic resonance data. Using modern structure validation criteria, including several formally adopted by the Protein Data Bank, and a clear measure of structural accuracy, the results show superior performance relative to previous Xplor-NIH implementations. Additionally, the Xplor-NIH structures compare favorably against originally determined NMR models.

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis-Trans Isomerization and the Chameleonic Roles of Calcium.

Falling outside of Lipinski's rule of five, macrocyclic drugs have accessed unique binding sites of their target receptors unreachable by traditional small molecules. Cyclosporin(e) A (CycA), an extensively studied macrocyclic natural product, is an immunosuppressant with undesirable side effects such as electrolytic imbalances. In this work, a comprehensive view on the conformational landscape of CycA, its interactions with Ca2+, and host-guest interactions with cyclophilin A (CypA) is reported through exhaustive analyses that combine ion-mobility spectrometry-mass spectrometry (IMS-MS), nuclear magnetic resonance (NMR) spectroscopy, distance-geometry modeling, and NMR-driven molecular dynamics. Our IMS-MS data show that CycA can adopt extremely compact conformations with significantly smaller collisional cross sections than the closed conformation observed in CDCl3. To adopt these conformations, the macrocyclic ring has to twist and bend via cis-trans isomerization of backbone amides, and thus, we termed this family of structures the "bent" conformation. Furthermore, NMR measurements indicate that the closed conformation exists at 19% in CD3OD/H2O and 55% in CD3CN. However, upon interacting with Ca2+, in addition to the bent and previously reported closed conformations of free CycA, the CycA:Ca2+ complex is open and has all-trans peptide bonds. Previous NMR studies using calcium perchlorate reported only the closed conformation of CycA (which contains one cis peptide bond). Here, calcium chloride, a more biologically relevant salt, was used, and interestingly, it helps converting the cis-MeLeu9-MeLeu10 peptide bond into a trans bond. Last, we were able to capture the native binding of CycA and CypA to give forth evidence that IMS-MS is able to probe the solution-phase structures of the complexes and that the Ca2+:CycA complex may play an essential role in the binding of CycA to CypA.

A three-dimensional potential of mean force to improve backbone and sidechain hydrogen bond geometry in Xplor-NIH protein structure determination.

We introduce a new hydrogen bonding potential of mean force generated from high-quality crystal structures for use in Xplor-NIH structure calculations. This term applies to hydrogen bonds involving both backbone and sidechain atoms. When used in structure refinement calculations of 10 example protein systems with experimental distance, dihedral and residual dipolar coupling restraints, we demonstrate that the new term has superior performance to the previously developed hydrogen bonding potential of mean force used in Xplor-NIH.

NMR Analysis of Apo Glutamine-Binding Protein Exposes Challenges in the Study of Interdomain Dynamics.

Glutamine-binding protein (GlnBP) displays an apo, "open" and a holo, "closed" crystal form, mutually related by a rigid-body reorientation of its domains. A fundamental question about such large-scale conformational transitions, whether the closed state exists in the absence of ligand, is controversial in the case of GlnBP. NMR observations have indicated no evidence of the closed form, whereas experimentally validated computations have suggested a remarkable ca. 40 % population. Herein, a paramagnetic NMR strategy designed to detect the putative apo-closed species shows that a major population of the latter is highly improbable. Further, NMR residual dipolar couplings collected under three anisotropic conditions do not reveal differential domain alignment and establish that the average solution conformation is satisfied by the apo-open crystal structure. Our results indicate that the computational prediction of large-scale interdomain motions is not trivial and may lead to erroneous conclusions without proper experimental validation.

Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein.

NMR has provided a wealth of structural and dynamical information for RNA molecules of up to ∼50 nucleotides, but its application to larger RNAs has been hampered in part by difficulties establishing global structural features. A potential solution involves measurement of NMR perturbations after site-specific paramagnetic labeling. Although the approach works well for proteins, the inability to place the label at specific sites has prevented its application to larger RNAs transcribed in vitro. Here, we present a strategy in which RNA loop residues are modified to promote binding to a paramagnetically tagged reporter protein. Lanthanide-induced pseudocontact shifts are demonstrated for a 232-nucleotide RNA bound to tagged derivatives of the spliceosomal U1A RNA-binding domain. Further, the method is validated with a 36-nucleotide RNA for which measured NMR values agreed with predictions based on the previously known protein and RNA structures. The ability to readily insert U1A binding sites into ubiquitous hairpin and/or loop structures should make this approach broadly applicable for the atomic-level study of large RNAs.

Protein Structure Elucidation from NMR Data with the Program Xplor-NIH.

Xplor-NIH is a popular software package for biomolecular structure determination from NMR (and other) experimental data. This chapter illustrates its use with the de novo structure determination of the B1 domain of streptococcal protein G (GB1), based on distances from nuclear Overhauser effects, torsion angles from scalar couplings, and bond-vector orientations from residual dipolar couplings. Including Xplor-NIH's latest developments, a complete structure calculation script is discussed in detail, and is intended to serve as a basis for other applications.

Xplor-NIH for molecular structure determination from NMR and other data sources.

Xplor-NIH is a popular software package for biomolecular structure determination from nuclear magnetic resonance (NMR) and other data sources. Here, some of Xplor-NIH's most useful data-associated energy terms are reviewed, including newer alternative options for using residual dipolar coupling data in structure calculations. Further, we discuss new developments in the implementation of strict symmetry for the calculation of symmetric homo-oligomers, and in the representation of the system as an ensemble of structures to account for motional effects. Finally, the different available force fields are presented, among other Xplor-NIH capabilities.

Improving NMR Structures of RNA.

Here, we show that modern solution nuclear magnetic resonance (NMR) structures of RNA exhibit more steric clashes and conformational ambiguities than their crystallographic X-ray counterparts. To tackle these issues, we developed RNA-ff1, a new force field for structure calculation with Xplor-NIH. Using seven published NMR datasets, RNA-ff1 improves covalent geometry and MolProbity validation criteria for clashes and backbone conformation in most cases, relative to both the previous Xplor-NIH force field and the original structures associated with the experimental data. In addition, with smaller base-pair step rises in helical stems, RNA-ff1 structures enjoy more favorable base stacking. Finally, structural accuracy improves in the majority of cases, as supported by complete residual dipolar coupling cross-validation. Thus, the reported advances show great promise in bridging the quality gap that separates NMR and X-ray structures of RNA.

Accurate high-throughput structure mapping and prediction with transition metal ion FRET.

Mapping the landscape of a protein's conformational space is essential to understanding its functions and regulation. The limitations of many structural methods have made this process challenging for most proteins. Here, we report that transition metal ion FRET (tmFRET) can be used in a rapid, highly parallel screen, to determine distances from multiple locations within a protein at extremely low concentrations. The distances generated through this screen for the protein maltose binding protein (MBP) match distances from the crystal structure to within a few angstroms. Furthermore, energy transfer accurately detects structural changes during ligand binding. Finally, fluorescence-derived distances can be used to guide molecular simulations to find low energy states. Our results open the door to rapid, accurate mapping and prediction of protein structures at low concentrations, in large complex systems, and in living cells.

Structural mechanism of Bax inhibition by cytomegalovirus protein vMIA.

The human protein Bax sits at a critical regulatory junction of apoptosis, or programmed cell death. Bax exists in equilibrium between cytosolic and mitochondria-associated forms that shifts toward the latter when Bax is activated by proapoptotic proteins. Activated Bax changes conformation, inserts into the mitochondrial outer membrane (MOM), oligomerizes, and induces MOM permeabilization, causing the release of cytochrome c, which effectively commits the cell to die. Because apoptosis is also a basic defense mechanism against invading pathogens, many viruses have developed counteractive measures. Such is the case of human cytomegalovirus, the replication of which hinges on vMIA (viral mitochondria-localized inhibitor of apoptosis), a virus-encoded protein with a unique, albeit poorly understood antiapoptotic activity by which it binds and recruits Bax to mitochondria. Here we show, via the structure determination of the complex between Bax and a peptide comprising vMIA's Bax-binding domain, that vMIA contacts Bax at a previously unknown regulatory site. Notably, using full-length vMIA, the structure is independently confirmed by assays in human cells that measure Bax subcellular localization and cytochrome c release. Mutants that disrupt key intermolecular interactions disfavor vMIA's mitochondrial recruitment of Bax, and increase cytochrome c release upon apoptosis induction. In a more stringent test, an engineered binding interface that achieves wild-type-like charge complementarity, although in a reversed fashion, recovers wild-type behavior. The structure suggests that by stabilizing key elements in Bax needed to unravel for its MOM insertion and oligomerization, vMIA prevents these important steps in apoptosis.

Smooth statistical torsion angle potential derived from a large conformational database via adaptive kernel density estimation improves the quality of NMR protein structures.

Statistical potentials that embody torsion angle probability densities in databases of high-quality X-ray protein structures supplement the incomplete structural information of experimental nuclear magnetic resonance (NMR) datasets. By biasing the conformational search during the course of structure calculation toward highly populated regions in the database, the resulting protein structures display better validation criteria and accuracy. Here, a new statistical torsion angle potential is developed using adaptive kernel density estimation to extract probability densities from a large database of more than 106 quality-filtered amino acid residues. Incorporated into the Xplor-NIH software package, the new implementation clearly outperforms an older potential, widely used in NMR structure elucidation, in that it exhibits simultaneously smoother and sharper energy surfaces, and results in protein structures with improved conformation, nonbonded atomic interactions, and accuracy.

Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N-15N and carbonyl 13C-13C dipolar recoupling data.

Recent structural studies of uniformly (15)N, (13)C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic (15)N and (13)C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole-dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole-dipole couplings among (15)N-labeled backbone amide sites and among (13)C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call (15)N-BARE and (13)C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. (15)N-BARE and (13)C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental (15)N and (13)C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the (15)N-BARE and (13)C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly (15)N, (13)C-labeled protein GB1. Results for GB1 show that (15)N-BARE and (13)C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures.

Solution NMR studies of periplasmic binding proteins and their interaction partners.

Periplasmic binding proteins (PBPs) are a crucial part of ATP-binding cassette import systems in Gram-negative bacteria. Central to their function is the ability to undergo a large-scale conformational rearrangement from open-unliganded to closed-liganded, which signals the presence of substrate and starts its translocation. Over the years, PBPs have been extensively studied not only owing to their essential role in nutrient uptake but also because they serve as excellent models for both practical applications (e.g., biosensor technology) and basic research (e.g., allosteric mechanisms). Although much of our knowledge at atomic level has been inferred from the detailed, static pictures afforded by crystallographic studies, nuclear magnetic resonance (NMR) has been able to fill certain gaps in such body of work, particularly with regard to dynamic processes. Here, we review NMR studies on PBPs, and their unique insights on conformation, dynamics, energetics, substrate binding, and interactions with related transport proteins. Based on the analysis of recent paramagnetic NMR results, as well as crystallographic and functional observations, we propose a mechanism that could explain the ability of certain PBPs to achieve a closed conformation in absence of ligand while others seem to remain open until ligand-mediated closure.

Ligand-free open-closed transitions of periplasmic binding proteins: the case of glutamine-binding protein.

The ability to undergo large-scale domain rearrangements is essential for the substrate-binding function of periplasmic binding proteins (PBPs), which are indispensable for nutrient uptake in Gram-negative bacteria. Crystal structures indicate that PBPs typically adopt either an "open" unliganded configuration or a "closed" liganded one. However, it is not clear whether, as a general rule, PBPs remain open until ligand-induced interdomain closure or are in equilibrium with a minor population of unliganded, closed species. Evidence for the latter has been recently reported on maltose-binding protein (MBP) in aqueous solution [Tang, C., et al. (2007) Nature 449, 1078-1082] via paramagnetic relaxation enhancement (PRE), a technique able to probe lowly populated regions of conformational space. Here, we use PRE to study the unliganded open-closed transition of another PBP: glutamine-binding protein (GlnBP). Through a combination of domain structure knowledge and intermolecular and concentration dependence PRE experiments, a set of surface residues was found to be involved in intermolecular interactions. Barring such residues, PRE data on ligand-free GlnBP, paramagnetically labeled at two sites (one at a time), could be appropriately explained by the unliganded, open crystal structure in that it both yielded a good PRE fit and was not significantly affected by PRE-based refinement. Thus, contrary to MBP, our data did not particularly suggest the coexistence of a minor closed conformer. Several possibilities were explored to explain the observed differences in such closely structurally related systems; among them, a particularly interesting one arises from close inspection of the interdomain "hinge" region of various PBPs: strong hydrogen bond interactions discourage large-scale interdomain dynamics.

Determination of the solution-bound conformation of an amino acid binding protein by NMR paramagnetic relaxation enhancement: use of a single flexible paramagnetic probe with improved estimation of its sampling space.

We demonstrate the feasibility of elucidating the bound ("closed") conformation of a periplasmic binding protein, the glutamine-binding protein (GlnBP), in solution, using paramagnetic relaxation enhancements (PREs) arising from a single paramagnetic group. GlnBP consists of two globular domains connected by a hinge. Using the ligand-free ("open") conformation as a starting point, conjoined rigid-body/torsion-angle simulated annealing calculations were performed using backbone (1)H(N)-PREs as a major source of distance information. Paramagnetic probe flexibility was accounted for via a multiple-conformer representation. A conventional approach where the entire PRE data set is enforced at once during simulated annealing yielded poor results due to inappropriate conformational sampling of the probe. On the other hand, significant improvements in coordinate accuracy were obtained by estimating the probe sampling space prior to structure calculation. Such sampling is achieved by refining the ensemble of probe conformers with intradomain PREs only, keeping the protein backbone fixed in the open form. Subsequently, while constraining the probe to the previously found conformations, the domains are allowed to move relative to each other under the influence of the non-intradomain PREs, giving the hinge region torsional degrees of freedom. Thus, by partitioning the protocol into "probe sampling" and "backbone sampling" stages, structures significantly closer to the X-ray structure of ligand-bound GlnBP were obtained.

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.