15N Relaxation Data Research Articles

B-box1 Domain of MID1 Interacts with the Ube2D1 E2 Enzyme Differently Than RING E3 Ligases.

The MID1 TRIM protein is important for ventral midline development in vertebrates, and mutations of its B-box1 domain result in several birth defects. The B-box1 domain of the human MID1 protein binds two zinc atoms and adopt a similar ββα-RING structure. This domain is required for the efficient ubiquitination of protein phosphatase 2A, alpha4, and fused kinase. Considering the structural similarity, the MID1 B-box1 domain exhibits mono-autoubiquitination activity, in contrast to poly-autoubiquitination observed for RING E3 ligases. To understand its mechanism of action, the interaction of the B-box1 domain with Ube2D1 (UbcH5a, E2), a preferred E2 ligase, is investigated. Using isothermal titration calorimetry, the MID1 RING and B-box1 domains were observed to have similar binding affinities with the Ube2D1 protein. However, NMR 15N-1H Heteronuclear Single Quantum Coherence titration, 15N relaxation data, and High Ambiguity Driven protein-protein DOCKing (HADDOCK) calculations show the B-box1 domain binding on a surface distinct from where RING domains bind. The novel binding interaction shows the B-box1 domain partially overlapping the noncovalent Ube2D1 and a ubiquitin binding site that is necessary for poly-autoubiquitination activity. The B-box1 domain also displaces the ubiquitin from the Ube2D1 protein. These studies reveal a novel binding interaction between the zinc-binding ββα-fold B-box1 domain and the Ube2D enzyme family and that this difference in binding, compared to RING E3 ligases, provides a rationale for its auto-monoubiquitination E3 ligase activity.

Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.

Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis.

NMR relaxation analysis of the mobile residues in globular proteins is sensitive to the form of the experimentally fitted internal autocorrelation function, which is used to represent that motion. Different order parameter representations can precisely fit the same set of 15N R1, R2, and heteronuclear NOE measurements while yielding significantly divergent predictions of the underlying autocorrelation functions, indicating the insufficiency of these experimental relaxation data for assessing which order parameter representation provides the most physically realistic predictions. Molecular dynamics simulations offer an unparalleled capability for discriminating among different order parameter representations to assess which representation can most accurately model a wide range of physically realistic autocorrelation functions. Six currently utilized AMBER and CHARMM force fields were applied to calculate autocorrelation functions for the backbone H–N bond vectors of ubiquitin as an operational test set. An optimized time constant-constrained triexponential (TCCT) representation was shown to markedly outperform the widely used (Sf2,τs,S2) extended Lipari–Szabo representation and the more closely related (Sf2,SH2, SN2) Larmor frequency-selective representation. Optimization of the TCCT representation at both 600 and 900 MHz 1H converged to the same parameterization. The higher magnetic field yielded systematically larger deviations in the back-prediction of the autocorrelation functions for the mobile amides, indicating little added benefit from multiple field measurements in analyzing amides that lack slower (∼ms) exchange line-broadening effects. Experimental 15N relaxation data efficiently distinguished among the different force fields with regard to their prediction of ubiquitin backbone conformational dynamics in the ps–ns time frame. While the earlier AMBER 99SB and CHARMM27 force fields underestimate the scale of backbone dynamics, which occur in this time frame, AMBER 14SB provided the most consistent predictions for the well-averaged highly mobile C-terminal residues of ubiquitin.

Accelerating 15N and 13C R1 and R1ρ relaxation measurements by multiple pathway solid-state NMR experiments

Magic angle spinning (MAS) Solid-state NMR is a powerful technique to probe dynamics of biological systems at atomic resolution. R1 and R1ρ relaxation measurements can provide detailed insight on amplitudes and time scales of motions, especially when information from several different site-specific types of probes is combined. However, such experiments are time-consuming to perform. Shortening the time necessary to record relaxation data for different nuclei will greatly enhance practicality of such approaches. Here, we present staggered acquisition experiments to acquire multiple relaxation experiments from a single excitation to reduce the overall experimental time. Our strategy enables one to collect 15N and 13C relaxation data in a single experiment in a fraction of the time necessary for two separate experiments, with the same signal to noise ratio.

Toho-1 β-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Backbone chemical shift assignments for the Toho-1 β-lactamase (263 amino acids, 28.9kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2H,13C,15N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-β-lactam β-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15N relaxation data at a single field of 16.4T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms of the macrodomain of SARS-CoV-2 non-structural protein 3b

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy.

Although the α-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a protein’s structure. Highly charged single α-helical (SAH) domains, consisting of a high percentage (>75%) of Arg, Lys, and Glu residues, are exceptions to this rule but have been difficult to characterize structurally. Our study focuses on the 68-residue medial tail domain of myosin-VI, which is found to contain a highly ordered α-helical structure extending from Glu-6 to Lys-63. High hydrogen exchange protection factors (15–150), small (ca. 4 Hz) 3JHNHα couplings, and a near-perfect fit to an ideal model α-helix for its residual dipolar couplings (RDCs), measured in a filamentous phage medium, support the high regularity of this helix. Remarkably, the hydrogen exchange rates are far more homogeneous than the protection factors derived from them, suggesting that for these transiently broken helices the intrinsic exchange rates derived from the amino acid sequence are not appropriate reference values. 15N relaxation data indicate a very high degree of rotational diffusion anisotropy (D∥/D⊥ ≈ 7.6), consistent with the hydrodynamic behavior predicted for such a long, nearly straight α-helix. Alignment of the helix by a paramagnetic lanthanide ion attached to its N-terminal region shows a decrease in alignment as the distance from the tagging site increases. This decrease yields a precise measure for the persistence length of 224 ± 10 Å at 20 °C, supporting the idea that the role of the SAH helix is to act as an extension of the myosin-VI lever arm.

NMR– and MD simulation–based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated

The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and 15N relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe3049 and Trp3052) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules.

Minute Additions of DMSO Affect Protein Dynamics Measurements by NMR Relaxation Experiments through Significant Changes in Solvent Viscosity.

Studies of protein−ligand binding often rely on dissolving the ligand in dimethyl sulfoxide (DMSO) to achieve sufficient solubility, and then titrating the ligand solution into the protein solution. As a result, the final protein−ligand solution contains small amounts of DMSO in the buffer. Here we report how the addition of DMSO impacts studies of protein conformational dynamics. We used 15N NMR relaxation to compare the rotational diffusion correlation time (τ C) of proteins in aqueous buffer with and without DMSO. We found that τ C scales with the viscosity of the water−DMSO mixture, which depends sensitively on the amount of DMSO and varies by a factor of 2 across the relevant concentration range. NMR relaxation studies of side chains dynamics are commonly interpreted using τ C as a fixed parameter, obtained from backbone 15N relaxation data acquired on a separate sample. Model‐free calculations show that errors in τ C, arising from mismatched DMSO concentration between samples, lead to significant errors in order parameters. Our results highlight the importance of determining τ C for each sample or carefully matching the DMSO concentrations between samples.

Fast evaluation of protein dynamics from deficient 15N relaxation data

Simple and convenient method of protein dynamics evaluation from the insufficient experimental 15N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse 15N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S2 and Rex, can be elucidated. The generalized order parameter, S2, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, Rex, identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.

NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na+ channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of 13C,15N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na+ channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn2+ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. 15N relaxation data revealed characteristic pattern of μs–ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps–ns time scale as compared to isolated VSDs of K+ channels. These results validate structural studies of isolated VSDs of Na+ channels and show possible pitfalls in application of this ‘divide and conquer’ approach.

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR 15N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR 15N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.

Chimeric Avidin – NMR Structure and Dynamics of a 56 kDa Homotetrameric Thermostable Protein

Chimeric avidin (ChiAVD) is a product of rational protein engineering remarkably resistant to heat and harsh conditions. In quest of the fundamentals behind factors affecting stability we have elucidated the solution NMR spectroscopic structure of the biotin–bound form of ChiAVD and characterized the protein dynamics through 15N relaxation and hydrogen/deuterium (H/D) exchange of this and the biotin–free form. To surmount the challenges arising from the very large size of the protein for NMR spectroscopy, we took advantage of its high thermostability. Conventional triple resonance experiments for fully protonated proteins combined with methyl–detection optimized experiments acquired at 58°C were adequate for the structure determination of this 56 kDa protein. The model–free parameters derived from the 15N relaxation data reveal a remarkably rigid protein at 58°C in both the biotin–bound and the free forms. The H/D exchange experiments indicate a notable increase in hydrogen protection upon biotin binding.

GP41 Ectodomain Dissociates and Forms a Stable Monomer on Phospholipid Vesicles and Detergent Micelles: Implication for the HIV-1 Env-Mediated Membrane Fusion

The first step of HIV infection involves the fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoproteins, gp120 and gp41. The binding of gp120 to the cell surface receptors CD4 triggers a cascade of conformational changes that disrupt the gp41-gp120 interactions and allows the insertion of the N-terminal fusion peptide of gp41 into the host cell membrane. The gp41 trimer then rapidly folds into a 6-helix bundle that pulls together the fusion peptide, inserted in the host cell membrane and the transmembrane domain, located in the viral membrane. Very little structural information is known about the pre-fusion state of gp41 notwithstanding its critical importance for the design of fusion inhibitors. To investigate the dynamics and structural properties of such metastable states, we designed a set of protein constructs mimicking the extracellular ectodomain of gp41. We found that the secondary structure of gp41 ectodomain is pronouncedly impacted by the presence of phospholipid vesicles or detergents containing phosphatidyl choline head groups. Our NMR and multi-angle light scattering data attribute these changes to the transition between a trimer in the absence of detergent to a monomer in the presence of a membrane-mimicking environment. The structure of gp41 in a monomeric state was determined by state-of-the-art NMR techniques, including measurements of NOE distance restraints and residual dipolar couplings in weakly aligned solutions. 15N relaxation data provided a detailed view at the backbone dynamics of both the trimeric and monomeric conformations. This stable monomeric state may represent a crucial structural intermediate facilitating both the gp41 conformational change during fusion and the local apposition of the viral and cellular membranes.

Intramolecular binding mode of the C-terminus ofEscherichia colisingle-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB–ssDNA complex. 15N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.

Proton-decoupled CPMG: A better experiment for measuring 15N R2 relaxation in disordered proteins

15N R2 relaxation is one of the most informative experiments for characterization of intrinsically disordered proteins (IDPs). Small changes in nitrogen R2 rates are often used to determine how IDPs respond to various biologically relevant perturbations such as point mutations, posttranslational modifications and weak ligand interactions. However collecting high-quality 15N relaxation data can be difficult. Of necessity, the samples of IDPs are often prepared with low protein concentration and the measurement time can be limited because of rapid sample degradation. Furthermore, due to hardware limitations standard experiments such as 15N spin-lock and CPMG can sample the relaxation decay only to ca. 150ms. This is much shorter than 15N T2 times in disordered proteins at or near physiological temperature. As a result, the sampling of relaxation decay profiles in these experiments is suboptimal, which further lowers the precision of the measurements.Here we report a new implementation of the proton-decoupled (PD) CPMG experiment which allows one to sample 15N R2 relaxation decay up to ca. 0.5–1s. The new experiment has been validated through comparison with the well-established spin-lock measurement. Using dilute samples of denatured ubiquitin, we have demonstrated that PD-CPMG produces up to 3-fold improvement in the precision of the data. It is expected that for intrinsically disordered proteins the gains may be even more substantial. We have also shown that this sequence has a number of favorable properties: (i) the spectra are recorded with narrow linewidth in nitrogen dimension; (ii) 15N offset correction is small and easy to calculate; (iii) the experiment is immune to various spurious effects arising from solvent exchange; (iv) the results are stable with respect to pulse miscalibration and rf field inhomogeneity; (v) with minimal change, the pulse sequence can also be used to measure R2 relaxation of 15Nε spins in arginine side chains. We anticipate that the new experiment will be a valuable addition to the NMR toolbox for studies of IDPs.

Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. OEP16 is a transmembrane amino acid selective transporter with a molecular mass of 16 kDa [1]. Here we report high-resolution NMR results for OEP16 solubilized in SDS detergent micelles. Three-dimensional NMR experiments were performed on U-15N, 13C- labeled and 80% perdeuterated protein to assign protein backbone resonances and obtain secondary structure information. To understand the protein dynamics and function 15N relaxation, amino acid titration, and 2H exchange experiments were performed. The chemical shift data for 15N, 1HN, 13CO, 13Cα and 13Cβ nuclei obtained from three-dimensional NMR experiments were used to assign 95% of the backbone amino acid residues. Chemical shift data were analyzed using the TALOS+ program to detect the secondary structure elements and to estimate 1H-15N bond orientational order parameters. Our analysis leads to three important conclusions: the tertiary structure of OEP16 involves four transmembrane helices connected by flexible loop regions, it is likely to be monomeric in SDS micelles, and the ligand binding sites could be located. The α-helix content calculated in this method (55%) fits well with previous measurements (50%) [1]. Our 15N relaxation data supports the predicted secondary structure and helps in estimating the isotropic global rotational correlation time (τm) [2]. The value of τm indicates that OEP16 is a monomer in SDS micelles. The 2H exchange data revealed solvent exposed regions in the protein that aid in understanding the tertiary structural folding of the protein. The present study provides a basis for a high resolution structural determination of this membrane protein in future NMR experiments. [1] D. Ni et al. (2011) Protein Expression Purif. 80, 157-168. [2] M. F. Brown (1982) J. Chem. Phys. 77, 1576-1599.

The Homotrimeric HIV-1 Viral Coat Protein GP41 is Highly Dynamic

A solution NMR study on the structure and dynamics of the homotrimeric gp41 complex, reconstituted in dodecyl phosphatidylcholine (DPC) micelles, is presented. Observed resonances were assigned to the fusion peptide (FP), N-terminal heptad repeat (NHR) and immuno-dominant loop region (IL), whereas the C-terminal heptad repeat (CHR) region, membrane proximal external region (MPER) and transmembrane helix (TM) remain completely invisible to solution NMR. 15N relaxation data reveal a high degree of intrinsic mobility: The a-helical fusion peptide (FP) exhibits high amplitude motion on the fast nanosecond time scale relative to the NHR region. The linker between NHR and CHR shows both fast and slow (microsecond) motions.Figure 1: Homotrimeric gp41 shows a high degree of intrinsic mobility. Our data are compatible with the protein switching on a microsecond time scale between the pre-hairpin intermediate, three-helical bundle state, and the late-fusion, anti-parallel six-helical bundle. MPER and TM are not shown since their conformation cannot be evaluated from the available solution NMR data. While MPER and TM are absent from the spectra, the FP shows the most intense resonances, which excludes a strong interaction between FP and TM region.View Large Image | View Hi-Res Image | Download PowerPoint Slide

NMR Structure and Dynamics of the Response Regulator Sma0114 from Sinorhizobium Meliloti

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution NMR structure and dynamics of Sma0114 from the bacterium Sinorhizobium meliloti, the first such characterization of a receiver domain from the HWE-kinase family of two-component systems. The structure of Sma0114 adopts a prototypical α5/β5 Rossman-fold but has features that set it apart from other receiver domains. The fourth β-strand of Sma0114 houses a PFxFATGY sequence motif, common to many HWE-kinase-associated receiver domains. This sequence motif in Sma0114 may substitute for the conserved Y-T coupling mechanism, which propagates conformational transitions in the 455 (α4-β5-α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, Sma0114 lacks the fourth α-helix of the consensus 455 face and 15N relaxation data show that it is replaced by a segment that is flexible on the ps-ns timescale. Secondary structure prediction of Sma0114 and other HWE-kinase-associated receiver domains suggests that the absence of helix α4 may be a conserved property of this family. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg2+ and Ca2+ that are required for phosphorylation, and exhibits μs-ms active site dynamics similar to other receiver domains. Taken together, our results suggest that Sma0114 has a conserved active site but differs from typical receiver domains in the structure of the 455 face that is used to effect signal transduction following activation.

Analysis of 15N-1H NMR relaxation in proteins by a combined experimental and molecular dynamics simulation approach: picosecond-nanosecond dynamics of the Rho GTPase binding domain of plexin-B1 in the dimeric state indicates allosteric pathways.

We investigate picosecond–nanosecond dynamics of the Rho-GTPase Binding Domain (RBD) of plexin-B1, which plays a key role in plexin-mediated cell signaling. Backbone 15N relaxation data of the dimeric RBD are analyzed with the model-free (MF) method, and with the slowly relaxing local structure/molecular dynamics (SRLS-MD) approach. Independent analysis of the MD trajectories, based on the MF paradigm, is also carried out. MF is a widely popular and simple method, SRLS is a general approach, and SRLS-MD is an integrated approach we developed recently. Corresponding parameters from the RBD dimer, a previously studied RBD monomer mutant, and the previously studied complex of the latter with the GTPase Rac1, are compared. The L2, L3, and L4 loops of the plexin-B1 RBD are involved in interactions with other plexin domains, GTPase binding, and RBD dimerization, respectively. Peptide groups in the loops of both the monomeric and dimeric RBD are found to experience weak and moderately asymmetric local ordering centered approximately at the C(i–1)(α)–C(i)(α) axes, and nanosecond backbone motion. Peptide groups in the α-helices and the β-strands of the dimer (the β-strands of the monomer) experience strong and highly asymmetric local ordering centered approximately at the C(i–1)(α)–C(i)(α) axes (N–H bonds). N–H fluctuations occur on the picosecond time scale. An allosteric pathway for GTPase binding, providing new insights into plexin function, is delineated.