Structural and dynamics characterization of norovirus protease

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.     

Conformational dynamics in loop swap mutants of homologous fibronectin type III domains

Fibronectin type III (FN-III) domains are autonomously folded modules found in a variety of multidomain proteins. The 10th FN-III domain from fibronectin (fnFN10) and the 3rd FN-III domain from tenascin-C (tnFN3) have 27% sequence identity and the same overall fold; however, the CC' loop has a different pattern of backbone hydrogen bonds and the FG loop is longer in fnFN10 compared to tnFN3. To examine the influence of length, sequence, and context in determining dynamical properties of loops, CC' and FG loops were swapped between fnFN10 and tnFN3 to generate four mutant proteins and backbone conformational dynamics on ps-ns and mus-ms timescales were characterized by solution (15)N-NMR spin relaxation spectroscopy. The grafted loops do not strongly perturb the properties of the protein scaffold; however, specific effects of the mutations are observed for amino acids that are proximal in space to the sites of mutation. The amino acid sequence primarily dictates conformational dynamics when the wild-type and grafted loop have the same length, but both sequence and context contribute to conformational dynamics when the loop lengths differ. The results suggest that changes in conformational dynamics of mutant proteins must be considered in both theoretical studies and protein design efforts.     

Dynamics of the Hck-SH3 domain: comparison of experiment with multiple molecular dynamics simulations

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.     

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from ¹⁵N relaxation and side-chain dynamics from ¹³C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Relaxation kinetics and the glassiness of native proteins: coupling of timescales

We provide evidence that the onset of functional dynamics of folded proteins with elevated temperatures is associated with the effective sampling of its energy landscape under physiological conditions. The analysis is based on data describing the relaxation phenomena governing the backbone dynamics of bovine pancreatic trypsin inhibitor derived from molecular dynamics simulations, previously reported by us. By representing the backbone dynamics of the folded protein by three distinct regimes, it is possible to decompose its seemingly complex dynamics, described by a stretch exponential decay of the backbone motions. Of these three regimes, one is associated with the slow timescales due to the activity along the envelope of the energy surface defining the folded protein. Another, with fast timescales, is due to the activity along the pockets decorating the folded-state envelope. The intermediate regime emerges at temperatures where jumps between the pockets become possible. It is at the temperature window where motions corresponding to all three timescales become operative that the protein becomes active.     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

Nanosecond Motions in Proteins Impose Bounds on the Timescale Distributions of Local Dynamics

We elucidate the physics of protein dynamical transition via 10-100-ns molecular dynamics simulations at temperatures spanning 160-300 K. By tracking the energy fluctuations, we show that the protein dynamical transition is marked by a crossover from nonstationary to stationary processes that underlie the dynamics of protein motions. A two-timescale function captures the nonexponential character of backbone structural relaxations. One timescale is attributed to the collective segmental motions and the other to local relaxations. The former is well defined by a single-exponential, nanosecond decay, operative at all temperatures. The latter is described by a set of processes that display a distribution of timescales. Although their average remains on the picosecond timescale, the distribution is markedly contracted at the onset of the transition. It is shown that the collective motions impose bounds on timescales spanned by local dynamical processes. The nonstationary character below the transition implicates the presence of a collection of substates whose interactions are restricted. At these temperatures, a wide distribution of local-motion timescales, extending beyond that of nanoseconds, is observed. At physiological temperatures, local motions are confined to timescales faster than nanoseconds. This relatively narrow window makes possible the appearance of multiple channels for the backbone dynamics to operate.     

Subpicosecond dynamics in nucleotides measured by spontaneous Raman spectroscopy

The band widths in Raman spectra are sensitive to dynamics active on a time scale from 0.1 to 10 ps. The band widths of nucleotide vibrations and their dependence on temperature, concentration, and structure are reported. From the experimental band widths and second moments, it is derived that the adenine vibrations at 725, 1336, 1480, and 1575 cm⁻¹, and the uracil vibration at 787 cm⁻¹, are in the fast modulation limit. The correlation times of the perturbations are faster than 0.4 ps. Thermal melting of the helical structure in polynucleotides results in larger band widths, due to an increase in vibrational dephasing and energy relaxation as a consequence of the increased interaction of the base moieties with the solvent molecules. The band width of the 725 cm⁻¹ adenine vibration is dependent on the type and structure of the backbone. It is found to be perturbed by movements of the sugar-phosphate moiety relative to the base. The band width of the 1575 cm⁻¹ adenine vibration is found to be sensitive to the base-pairing interaction. From a comparison of the band widths in polynucleotides with a different base sequence (homopolymer vs alternating purine-pyrimidine sequence), it is concluded that resonant vibrational energy transfer between the base molecules is not important as a relaxation process for the vibrational band widths of nucleotides. Several theoretical models for the interpretation of band widths are discussed. The theory does not take into account the strong hydrogen-bonding nature water and hence fails to describe the observations in nucleotide-water systems. The bands of the carbonyl stretching vibrations are inhomogeneously broadened. The carbonyl groups have a strong dipolar interaction with the polar water molecules and are therefore strongly perturbed by coupling to the heatbath via hydrogen bonds. © 1997 John Wiley & Sons, Inc. Biopoly 41: 751–763, 1997     

Dynamics of vibrational frequency fluctuations in deuterated liquid ammonia: roles of fluctuating hydrogen bonds and free ND modes

We present an ab initio molecular dynamics study of the roles of fluctuating hydrogen bonds and free ND modes in the dynamics of ND stretch frequency fluctuations in deuterated liquid ammonia. We have also looked at some of the other dynamical quantities such as diffusion and orientational relaxation and also structural quantities such as pair correlations and hydrogen bonding properties which are relevant in the current context. The time correlation function of ND stretch frequencies is found to decay with primarily two time scales: A short-time decay with a time scale of less than 100 fs arising from intermolecular motion of intact hydrogen bonds and also from fast hydrogen bond breaking and a longer time scale of about 500 fs which can be assigned to the lifetime of free ND modes. Unlike water, in liquid ammonia an ND mode is found to remain free for a longer period than it stays hydrogen bonded and this longer lifetime of free ND modes determines the long-time behaviour of frequency fluctuations. Our hole dynamics calculations produced results of vibrational spectral diffusion that are similar to the decay of frequency time correlation. Inclusion of dispersion corrections is found to make the dynamics slightly faster.     

Backbone dynamics of the 8 kDa dynein light chain dimer reveals molecular basis of the protein's functional diversity

Axonemal and cytoplasmic dyneins share a highly conserved 8 kDa light chain (DLC8) for motor assembly and function. Other than serving as a light chain of dynein complexes, DLC8 has been shown to bind a larger number of proteins with diverse biological functions including cell cycle control, apoptosis, and cell polarity maintenance. Therefore, DLC8 is likely a multifunctional regulatory protein. DLC8 exists as a dimer in solution, and the protein dimer is capable of binding to two target molecules. In this work, the backbone dynamics of DLC8, both in its apo- and target-peptide bound forms, were characterized by ¹⁵N NMR relaxation studies. The relaxation data were analyzed using model-free approach. We show that the target peptide-binding region of apo-DLC8 experiences microsecond-to-millisecond time scale conformational fluctuation, suggesting that the target-binding region of the protein is capable of adjusting its shape and size in responding to its various targets. The conformational breathing of the target-binding region of apo-DLC8 was also supported by backbone amide exchange experiment. Such segmental conformational motion of the protein is significantly reduced upon forming a complex with a target peptide. The dynamic properties of DLC8 in solution provide insight into the protein's diverse sequence-dependent target binding.     

Solution conformation and dynamics of a fungal cell wall polysaccharide isolated from Microsporum gypseum

The conformational and dynamical features of a branched mannan isolated from a fungal cell wall have been analysed by homo and heteronuclear NMR methods, employing different magnetic fields. 1HNMR cross relaxation times have been obtained for this polysaccharide and have been interpreted qualitatively using different motional models. 13C NMR relaxation parameters (T1, T2, NOE) have also been measured and interpreted using different approximations based on the Lipari and Szabo model free approach. The analysis of the data indicate the existence of important flexibility for the different linkages of the polysaccharide. Motions in the range of 4–6 ns contribute to the relaxation of the macromolecule, although faster internal motions in the 500 ps and 100 ps timescales are also present. These time scales indicate that segmental motions as well as internal motions around the glycosidic linkages are the major sources of relaxation for this molecule at 318 K. Molecular dynamics simulations have also been performed. The obtained results also indicate that the polysaccharide possess a substantial amount of conformational freedom.     

Motional timescale predictions by molecular dynamics simulations: Case study using proline and hydroxyproline sidechain dynamics

We propose a new approach for force field optimizations which aims at reproducing dynamics characteristics using biomolecular MD simulations, in addition to improved prediction of motionally averaged structural properties available from experiment. As the source of experimental data for dynamics fittings, we use ¹³C NMR spin‐lattice relaxation times T₁ of backbone and sidechain carbons, which allow to determine correlation times of both overall molecular and intramolecular motions. For structural fittings, we use motionally averaged experimental values of NMR J couplings. The proline residue and its derivative 4‐hydroxyproline with relatively simple cyclic structure and sidechain dynamics were chosen for the assessment of the new approach in this work. Initially, grid search and simplexed MD simulations identified large number of parameter sets which fit equally well experimental J couplings. Using the Arrhenius‐type relationship between the force constant and the correlation time, the available MD data for a series of parameter sets were analyzed to predict the value of the force constant that best reproduces experimental timescale of the sidechain dynamics. Verification of the new force‐field (termed as AMBER99SB‐ILDNP) against NMR J couplings and correlation times showed consistent and significant improvements compared to the original force field in reproducing both structural and dynamics properties. The results suggest that matching experimental timescales of motions together with motionally averaged characteristics is the valid approach for force field parameter optimization. Such a comprehensive approach is not restricted to cyclic residues and can be extended to other amino acid residues, as well as to the backbone. Proteins 2014; 82:195–215. © 2013 Wiley Periodicals, Inc.     

Thermodynamic interpretation of protein dynamics from NMR relaxation measurements

Protein dynamics and thermodynamics can be characterized through measurements of relaxation rates of side chain (2)H and (13)C, and backbone (15)N nuclei using NMR spectroscopy. The rates reflect protein motions on timescales from picoseconds to milliseconds. Backbone and methyl side chain NMR relaxation measurements for several proteins are beginning to reveal the role of protein dynamics in protein stability and ligand binding.     

Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena.

Oxidized flavodoxin from Cyanobacterium anabaena PCC 7119 is used as a model system to investigate the fast internal dynamics of a flavin-bearing protein. Virtually complete backbone and side chain resonance NMR assignments of an oxidized flavodoxin point mutant (C55A) have been determined. Backbone and side chain dynamics in flavodoxin (C55A) were investigated using (15)N amide and deuterium methyl NMR relaxation methods. The squared generalized order parameters (S(NH)(2)) for backbone amide N-H bonds are found to be uniformly high ( approximately 0.923 over 109 residues in regular secondary structure), indicating considerable restriction of motion in the backbone of the protein. In contrast, methyl-bearing side chains are considerably heterogeneous in their amplitude of motion, as indicated by obtained symmetry axis squared generalized order parameters (S(axis)(2)). However, in comparison to nonprosthetic group-bearing proteins studied with these NMR relaxation methods, the side chains of oxidized flavodoxin are unusually rigid.     

Model selection for the interpretation of protein side chain methyl dynamics

A number of different dynamics models are considered for fitting ¹³C and ²H side chain methyl relaxation rates. It is shown that in cases where nanosecond time scale dynamics are present the extended Lipari–Szabo model which is explicitly parameterized to include the effects of slow motions can produce wide distributions of fitting parameters even in cases where the errors are relatively small and large numbers of relaxation rates are considered. In contrast, fits of ¹⁵N backbone dynamics using this model are far more robust. The origin of this difference is analyzed and can be explained by the different functional forms of the spectral density in these two cases. The utility of a number of models for the analysis of methyl side chain dynamics is presented.     

Phosphorylation-induced changes in backbone dynamics of the dematin headpiece C-terminal domain

Dematin is an actin-binding protein abundant in red blood cells and other tissues. It contains a villin-type ‘headpiece’ F-actin-binding domain at its extreme C-terminus. The isolated dematin headpiece domain (DHP) undergoes a significant conformational change upon phosphorylation. The mutation of Ser74 to Glu closely mimics the phosphorylation of DHP. We investigated motions in the backbone of DHP and its mutant DHPS74E using several complementary NMR relaxation techniques: laboratory frame ¹⁵N NMR relaxation, which is sensitive primarily to the ps–ns time scale, cross-correlated chemical shift modulation NMR relaxation detecting correlated μs–ms time scale motions of neighboring ¹³C′ and ¹⁵N nuclei, and cross-correlated relaxation of two ¹⁵N–¹H dipole–dipole interactions detecting slow motions of backbone NH vectors in successive amino acid residues. The results indicate a reduction in mobility upon the mutation in several regions of the protein. The additional salt bridge formed in DHPS74E that links the N- and C-terminal subdomains is likely to be responsible for these changes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Relating side-chain mobility in proteins to rotameric transitions: Insights from molecular dynamics simulations and NMR

The dynamic aspect of proteins is fundamental to understanding protein stability and function. One of the goals of NMR studies of side-chain dynamics in proteins is to relate spin relaxation rates to discrete conformational states and the timescales of interconversion between those states. Reported here is a physical analysis of side-chain dynamics that occur on a timescale commensurate with monitoring by ²H spin relaxation within methyl groups. Motivated by observations made from tens-of-nanoseconds long MD simulations on the small protein eglin c in explicit solvent, we propose a simple molecular mechanics-based model for the motions of side-chain methyl groups. By using a Boltzmann distribution within rotamers, and by considering the transitions between different rotamer states, the model semi-quantitatively correlates the population of rotamer states with ‘model-free’ order parameters typically fitted from NMR relaxation experiments. Two easy-to-use, analytical expressions are given for converting S²_axis’ values (order parameter for C–CH₃ bond) into side-chain rotamer populations. These predict that S²_axis’ values below 0.8 result from population of more than one rotameric state. The relations are shown to predict rotameric sampling with reasonable accuracy on the ps–ns timescale for eglin c and are validated for longer timescales on ubiquitin, for which side-chain residual dipolar coupling (RDC) data have been collected.     

Accurate and efficient description of protein vibrational dynamics: comparing molecular dynamics and Gaussian models

Current all-atom potential based molecular dynamics (MD) allows the identification of a protein's functional motions on a wide-range of timescales, up to few tens of nanoseconds. However, functional, large-scale motions of proteins may occur on a timescale currently not accessible by all-atom potential based MD. To avoid the massive computational effort required by this approach, several simplified schemes have been introduced. One of the most satisfactory is the Gaussian network approach based on the energy expansion in terms of the deviation of the protein backbone from its native configuration. Here, we consider an extension of this model that captures in a more realistic way the distribution of native interactions due to the introduction of effective side-chain centroids. Since their location is entirely determined by the protein backbone, the model is amenable to the same exact and computationally efficient treatment as previous simpler models. The ability of the model to describe the correlated motion of protein residues in thermodynamic equilibrium is established through a series of successful comparisons with an extensive (14 ns) MD simulation based on the AMBER potential of HIV-1 protease in complex with a peptide substrate. Thus, the model presented here emerges as a powerful tool to provide preliminary, fast yet accurate characterizations of protein near-native motion.