Residual Dipolar Coupling Data Research Articles

The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin

Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdHN2N3, A326–D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.

Visualizing the Inter-Domain Motions of a Pathogenic Protein using Sparse RDC Data

SpA-N is the N-terminal half of Staphylococcal protein A and it is composed of five protein binding domains. The five domains could bind to antibody, TNFR1 and von Willebrand factor and facilitate the evasion of Staphylococcus aureus into the human immune system. The functional plasticity is suggested to be the result of structural flexibility. Heteronuclear spin relaxation experiments demonstrated that the five domains of SpA-N are connected by four flexible linkers. In order to obtain additional dynamic information, we constructed a di-domain mimic of SpA-N with a lanthanide binding tag and measured residual dipolar couplings (RDCs). The di-domain construct can bind lanthanide ions and consequently be aligned in the presence of a strong magnetic field. By using different combinations of lanthanide ions and protein constructs, we obtained two orthogonal alignments, which have enough information content to determine a model with maximally 10 parameters. In addition, we designed a de novo method to extract dynamic information from RDCs. Instead of determining conventional structure ensembles, our method determines an inter-domain orientation distribution to describe the structure of a flexible protein using continuous probability distributions. By using continuous models, orientation distributions can be parameterized by a small number of variables and still remain general enough to describe a broad spectrum of inter-domain motions. As a result, the method can avoid the over-fitting problem even with sparse data. Because no force field is applied, the generated distribution is purely determined by RDC data and least biased. Using the method, we determined the inter-domain orientation distribution of the di-domain construct with only two orthogonal alignments. A strong correlation was observed in the distribution and conformations were well populated in a limited region.

Death Effector Domain Flexibility in Mediating Protein-Protein Interactions

PED/PEA-15 (phosphoprotein enriched in diabetes/astrocytes, 15 kD) is a small, non-catalytic, death-effector domain (DED) containing protein, that is widely expressed in different tissues and highly conserved among mammals. PED/PEA-15 has been found to interact with several protein targets in various pathways, including FADD and procaspase-8 (apoptosis), ERK1/2 (cell cycle entry), and PLD1/2 (diabetes). We have previously reported a surprising conformational change of PED/PEA-15 DED upon interaction with ERK2 using NMR dynamics and residual dipolar coupling (RDC) data. In the complex, PED/PEA-15 utilizes helices α1, α5, and α6 of the DED, in addition to the C-terminal tail, to binding to ERK2, while helices α2, α3, and α4 are highly flexible in the complex, and adopt a distinct relative orientation comparing to the free-form conformation. We have additionally modeled the PED/PEA-15 conformations in the ERK2 complex using CS/RDC-Rosetta protocol. Based on our NMR model of PED/PEA-15 complex with ERK2, we propose that the conserved charge triad motif on DED surface, comprising of D19-R72-D74L, which is located at a hinge position between these two dynamically distinct segments, mediates the necessary conformational changes to accommodate ERK2 binding. We believe that various conformations of the DED, facilitated by large number of surface polar interactions, may attribute to its ability to interact with structurally and functionally diverse proteins.

Conformation of inhibitor-free HIV-1 protease derived from NMR spectroscopy in a weakly oriented solution.

Flexibility of the glycine-rich flaps is known to be essential for catalytic activity of the HIV-1 protease, but their exact conformations at the different stages of the enzymatic pathway remain subject to much debate. Although hundreds of crystal structures of protease-inhibitor complexes have been solved, only about a dozen inhibitor-free protease structures have been reported. These latter structures reveal a large diversity of flap conformations, ranging from closed to semi-open to wide open. To evaluate the average structure in solution, we measured residual dipolar couplings (RDCs) and compared these to values calculated for crystal structures representative of the closed, semi-open, and wide-open states. The RDC data clearly indicate that the inhibitor-free protease, on average, adopts a closed conformation in solution that is very similar to the inhibitor-bound state. By contrast, a highly drug-resistant protease mutant, PR20, adopts the wide-open flap conformation.

FANTEN: a new web-based interface for the analysis of magnetic anisotropy-induced NMR data.

Pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) arising from the presence of paramagnetic metal ions in proteins as well as RDCs due to partial orientation induced by external orienting media are nowadays routinely measured as a part of the NMR characterization of biologically relevant systems. PCSs and RDCs are becoming more and more popular as restraints (1) to determine and/or refine protein structures in solution, (2) to monitor the extent of conformational heterogeneity in systems composed of rigid domains which can reorient with respect to one another, and (3) to obtain structural information in protein-protein complexes. The use of both PCSs and RDCs proceeds through the determination of the anisotropy tensors which are at the origin of these NMR observables. A new user-friendly web tool, called FANTEN (Finding ANisotropy TENsors), has been developed for the determination of the anisotropy tensors related to PCSs and RDCs and has been made freely available through the WeNMR ( http://fanten-enmr.cerm.unifi.it:8080 ) gateway. The program has many new features not available in other existing programs, among which the possibility of a joint analysis of several sets of PCS and RDC data and the possibility to perform rigid body minimizations.

Characterizing RNA ensembles from NMR data with kinematic models.

Functional mechanisms of biomolecules often manifest themselves precisely in transient conformational substates. Researchers have long sought to structurally characterize dynamic processes in non-coding RNA, combining experimental data with computer algorithms. However, adequate exploration of conformational space for these highly dynamic molecules, starting from static crystal structures, remains challenging. Here, we report a new conformational sampling procedure, KGSrna, which can efficiently probe the native ensemble of RNA molecules in solution. We found that KGSrna ensembles accurately represent the conformational landscapes of 3D RNA encoded by NMR proton chemical shifts. KGSrna resolves motionally averaged NMR data into structural contributions; when coupled with residual dipolar coupling data, a KGSrna ensemble revealed a previously uncharacterized transient excited state of the HIV-1 trans-activation response element stem–loop. Ensemble-based interpretations of averaged data can aid in formulating and testing dynamic, motion-based hypotheses of functional mechanisms in RNAs with broad implications for RNA engineering and therapeutic intervention.

Structural dynamics of a single-stranded RNA-helix junction using NMR.

Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3' end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, (13)C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A6-tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end. Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Our results reveal a surprising degree of structural and dynamic complexity at the ssRNA-helix junction, which involves a fine balance between order and disorder that may facilitate efficient pseudoknot formation on ligand recognition.

Solution structure of lysine‐free (K0) ubiquitin

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub (15) N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations.

DYNAFOLD: A DYNAMIC PROGRAMMING APPROACH TO PROTEIN BACKBONE STRUCTURE DETERMINATION FROM MINIMAL SETS OF RESIDUAL DIPOLAR COUPLINGS

Residual Dipolar Couplings (RDCs) are a source of NMR data that can provide a powerful set of constraints on the orientation of inter-nuclear vectors, and are quickly becoming a larger part of the experimental toolset for molecular biologists. However, few reliable protocols exist for the determination of protein backbone structures from small sets of RDCs. DynaFold is a new dynamic programming algorithm designed specifically for this task, using minimal sets of RDCs collected in multiple alignment media. DynaFold was first tested utilizing synthetic data generated for the N--H , C(α)--H(α), and C--N vectors of 1BRF, 1F53, 110M, and 3LAY proteins, with up to ±1 Hz error in three alignment media, and was able to produce structures with less than 1.9 Å of the original structures. DynaFold was then tested using experimental data, obtained from the Biological Magnetic Resonance Bank, for proteins PDBID:1P7E and 1D3Z using RDC data from two alignment media. This exercise yielded structures within 1.0 Å of their respective published structures in segments with high data density, and less than 1.9 Å over the entire protein. The same sets of RDC data were also used in comparisons with traditional methods for analysis of RDCs, which failed to match the accuracy of DynaFold's approach to structure determination.

Substantial Conformational Change Mediated by Charge-Triad Residues of the Death Effector Domain in Protein-Protein Interactions

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

Recovering a Representative Conformational Ensemble from Underdetermined Macromolecular Structural Data

Structural analysis of proteins and nucleic acids is complicated by their inherent flexibility, conferred, for example, by linkers between their contiguous domains. Therefore, the macromolecule needs to be represented by an ensemble of conformations instead of a single conformation. Determining this ensemble is challenging because the experimental data are a convoluted average of contributions from multiple conformations. As the number of the ensemble degrees of freedom generally greatly exceeds the number of independent observables, directly deconvolving experimental data into a representative ensemble is an ill-posed problem. Recent developments in sparse approximations and compressive sensing have demonstrated that useful information can be recovered from underdetermined (ill-posed) systems of linear equations by using sparsity regularization. Inspired by these advances, we designed the Sparse Ensemble Selection (SES) method for recovering multiple conformations from a limited number of observations. SES is more general and accurate than previously published minimum-ensemble methods, and we use it to obtain representative conformational ensembles of Lys48-linked diubiquitin, characterized by the residual dipolar coupling data measured at several pH conditions. These representative ensembles are validated against NMR chemical shift perturbation data and compared to maximum-entropy results. The SES method reproduced and quantified the previously observed pH dependence of the major conformation of Lys48-linked diubiquitin, and revealed lesser-populated conformations that are preorganized for binding known diubiquitin receptors, thus providing insights into possible mechanisms of receptor recognition by polyubiquitin. SES is applicable to any experimental observables that can be expressed as a weighted linear combination of data for individual states.

BCL::Fold—Protein topology determination from limited NMR restraints

When experimental protein NMR data are too sparse to apply traditional structure determination techniques, de novo protein structure prediction methods can be leveraged. Here, we describe the incorporation of NMR restraints into the protein structure prediction algorithm BCL::Fold. The method assembles discreet secondary structure elements using a Monte Carlo sampling algorithm with a consensus knowledge-based energy function. New components were introduced into the energy function to accommodate chemical shift, nuclear Overhauser effect, and residual dipolar coupling data. In particular, since side chains are not explicitly modeled during the minimization process, a knowledge based potential was created to relate experimental side chain proton-proton distances to Cβ -Cβ distances. In a benchmark test of 67 proteins of known structure with the incorporation of sparse NMR restraints, the correct topology was sampled in 65 cases, with an average best model RMSD100 of 3.4 ± 1.3 Å versus 6.0 ± 2.0 Å produced with the de novo method. Additionally, the correct topology is present in the best scoring 1% of models in 61 cases. The benchmark set includes both soluble and membrane proteins with up to 565 residues, indicating the method is robust and applicable to large and membrane proteins that are less likely to produce rich NMR datasets.

Conformational Analysis of the Anti‐obesity Drug Lorcaserin in Water: How To Take Advantage of Long‐Range Residual Dipolar Couplings

The conformational state of 8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (lorcaserin) in water has been determined on the basis of one-bond and long-range C-H residual dipolar coupling (RDC) data along with DFT computations and (3)J(HH) coupling-constant analysis. According to this analysis, lorcaserin exists as a conformational equilibrium of two crown-chair forms, of which the preferred conformation has the methyl group in an equatorial orientation.

ORIUM: Optimized RDC-based Iterative and Unified Model-free analysis

Residual dipolar couplings (RDCs) are NMR parameters that provide both structural and dynamic information concerning inter-nuclear vectors, such as N–HN and Cα–Hα bonds within the protein backbone. Two approaches for extracting this information from RDCs are the model free analysis (MFA) (Meiler et al. in J Am Chem Soc 123:6098–6107, 2001; Peti et al. in J Am Chem Soc 124:5822–5833, 2002) and the direct interpretation of dipolar couplings (DIDCs) (Tolman in J Am Chem Soc 124:12020–12030, 2002). Both methods have been incorporated into iterative schemes, namely the self-consistent RDC based MFA (SCRM) (Lakomek et al. in J Biomol NMR 41:139–155, 2008) and iterative DIDC (Yao et al. in J Phys Chem B 112:6045–6056, 2008), with the goal of removing the influence of structural noise in the MFA and DIDC formulations. Here, we report a new iterative procedure entitled Optimized RDC-based Iterative and Unified Model-free analysis (ORIUM). ORIUM unifies theoretical concepts developed in the MFA, SCRM, and DIDC methods to construct a computationally less demanding approach to determine these structural and dynamic parameters. In all schemes, dynamic averaging reduces the actual magnitude of the alignment tensors complicating the determination of the absolute values for the generalized order parameters. To readdress this scaling issue that has been previously investigated (Lakomek et al. in J Biomol NMR 41:139–155, 2008; Salmon et al. in Angew Chem Int Edit 48:4154–4157, 2009), a new method is presented using only RDC data to establish a lower bound on protein motion, bypassing the requirement of Lipari–Szabo order parameters. ORIUM and the new scaling procedure are applied to the proteins ubiquitin and the third immunoglobulin domain of protein G (GB3). Our results indicate good agreement with the SCRM and iterative DIDC approaches and signify the general applicability of ORIUM and the proposed scaling for the extraction of inter-nuclear vector structural and dynamic content.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-013-9775-1) contains supplementary material, which is available to authorized users.

Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

Protein Structure Validation and Identification from Unassigned Residual Dipolar Coupling Data Using 2D-PDPA

More than 90% of protein structures submitted to the PDB each year are homologous to some previously characterized protein structure. The extensive resources that are required for structural characterization of proteins can be justified for the 10% of the novel structures, but not for the remaining 90%. This report presents the 2D-PDPA method, which utilizes unassigned residual dipolar coupling in order to address the economics of structure determination of routine proteins by reducing the data acquisition and processing time. 2D-PDPA has been demonstrated to successfully identify the correct structure of an array of proteins that range from 46 to 445 residues in size from a library of 619 decoy structures by using unassigned simulated RDC data. When using experimental data, 2D-PDPA successfully identified the correct NMR structures from the same library of decoy structures. In addition, the most homologous X-ray structure was also identified as the second best structural candidate. Finally, success of 2D-PDPA in identifying and evaluating the most appropriate structure from a set of computationally predicted structures in the case of a previously uncharacterized protein Pf2048.1 has been demonstrated. This protein exhibits less than 20% sequence identity to any protein with known structure and therefore presents a compelling and practical application of our proposed work.

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.

The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

(15) N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H(N) -H(α) coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (λ lysozyme) in solution. The lower and upper lip regions in λ lysozyme (residues 51-60 and 128-141, respectively) show reduced (1) H-(15) N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T2 indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for λ lysozyme indicate that two fluctuating β-strands (β3 and β4) are populated in the lower lip region while the N terminus of helix α6 (residues 136-139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the β-sheet and helical secondary structure in the lip regions, with populations of 40-60 %. Thus in solution λ lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein.

A General Method for Constructing Atomic-Resolution RNA Ensembles using NMR Residual Dipolar Couplings: The Basis for Interhelical Motions Revealed

The ability to modulate alignment and measure multiple independent sets of NMR residual dipolar couplings (RDCs) has made it possible to characterize internal motions in proteins at atomic resolution and with time scale sensitivity ranging from picoseconds up to milliseconds. The application of such methods to the study of RNA dynamics, however, remains fundamentally limited by the inability to modulate alignment and by strong couplings between internal and overall motions that complicate the quantitative interpretation of RDCs. Here, we address this problem by showing that RNA alignment can be generally modulated, in a controlled manner, by variable elongation of A-form helices and that the information contained within the measured RDCs can be extracted even in the presence of strong couplings between motions and overall alignment via structure-based prediction of alignment. Using this approach, four RDC data sets, and a broad conformational pool obtained from a 8.2 μs molecular dynamics simulation, we successfully construct and validate an atomic resolution ensemble of human immunodeficiency virus type I transactivation response element RNA. This ensemble reveals local motions in and around the bulge involving changes in stacking and hydrogen-bonding interactions, which are undetectable by traditional spin relaxation and drive global changes in interhelical orientation. This new approach broadens the scope of using RDCs in characterizing the dynamics of nucleic acids.

Chiral discrimination of amines by anisotropic NMR parameters using chiral polyacrylamide‐based gels

A new chiral alignment medium for dimethyl sulfoxide, methanol, and water as solvents was developed. Because both enantiomers of the gel are available, it is possible to enantiodiscriminate natural products such as strychnine HCl that naturally occurs as single enantiomer. With the two methods of achieving anisotropy, namely stretching and confinement, the degree of alignment can be adjusted, and the director changed from horizontal to vertical. This increases the applicability. Three compounds were enantiodiscriminated on the basis of residual dipolar coupling data: mefloquine HCl, strychnine HCl, and menthylamine HCl.