Correlation between local structural dynamics of proteins inferred from NMR ensembles and evolutionary dynamics of homologues of known structure

Conformational changes in proteins are extremely important for their biochemical functions. Correlation between inherent conformational variations in a protein and conformational differences in its homologues of known structure is still unclear. In this study, we have used a structural alphabet called Protein Blocks (PBs). PBs are used to perform abstraction of protein 3-D structures into a 1-D strings of 16 alphabets (a–p) based on dihedral angles of overlapping pentapeptides. We have analyzed the variations in local conformations in terms of PBs represented in the ensembles of 801 protein structures determined using NMR spectroscopy. In the analysis of concatenated data over all the residues in all the NMR ensembles, we observe that the overall nature of inherent local structural variations in NMR ensembles is similar to the nature of local structural differences in homologous proteins with a high correlation coefficient of .94. High correlation at the alignment positions corresponding to helical and β-sheet regions is only expected. However, the correlation coefficient by considering only the loop regions is also quite high (.91). Surprisingly, segregated position-wise analysis shows that this high correlation does not hold true to loop regions at the structurally equivalent positions in NMR ensembles and their homologues of known structure. This suggests that the general nature of local structural changes is unique; however most of the local structural variations in loop regions of NMR ensembles do not correlate to their local structural differences at structurally equivalent positions in homologues.     

Protein local conformations arise from a mixture of Gaussian distributions

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures, which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF’s corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end, we have analyzed the correlation between sequence features and the conformational classes.     

Molecular Mechanics and Dynamics of an Abasic Frameshift in DNA and Comparison to NMR Data

Abstract

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide:

5′d(C1 P1 G2 P2 G3 P3 dr4 P4 G5 P5 G6 P6 C7)3′

3′d(G13P12C12PllCll P10 C10 P9 C9 P8 G8)5′

where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2′ endo or C3′ endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4.

In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium.

Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

     

Global and Local Structural Properties of the Principal Neutralizing Determinant of the HIV-1 Envelope Protein gp120

Abstract

The model of spatial structure for the principal neutralizing determinant (PND) of the HIV-1 envelope protein gpl20 is proposed in terms of two-dimensional nuclear Overhauser effect (NOE) spectroscopy data. To build the model, the NMR-based theoretical conformational analysis of synthetic PND peptides of length 40, 24, and 12 residues is carried out. The modeling of the molecular spatial structures is performed by a new approach to research of conformationally mobile peptides using the algorithms of the restrained molecular mechanics method developed earlier. The following major conclusions are made based on the analysis of the simulated peptide conformations: i) there is not unique PND structure in solution, ii) there are seven different PND structures each of which agrees with the experimental data and stereochemical criteria used in computing its spatial model, iii) the PND is characterized by irregular conformation containing a number of reverse turns, iv) all of the selected conformations are conserved in the Gly-Pro-Gly-Arg-Ala-Phe stretch, the most provable viral immunodominant epitope. These data allow to suppose that binding properties of this site are determined by the structural motif which forms the conformation of a double β-turn and appears common for all hexapeptide structures.     

Non-B DNA conformations analysis through molecular dynamics simulations

BackgroundNon-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded in a hot spot for the 3′ end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome.MethodsTo better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated.ResultsOur results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages.ConclusionsWe showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations.General significanceThe fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.     

A structural alphabet for local protein structures: improved prediction methods

Three-dimensional protein structures can be described with a library of 3D fragments that define a structural alphabet. We have previously proposed such an alphabet, composed of 16 patterns of five consecutive amino acids, called Protein Blocks (PBs). These PBs have been used to describe protein backbones and to predict local structures from protein sequences. The Q16 prediction rate reaches 40.7% with an optimization procedure. This article examines two aspects of PBs. First, we determine the effect of the enlargement of databanks on their definition. The results show that the geometrical features of the different PBs are preserved (local RMSD value equal to 0.41 A on average) and sequence-structure specificities reinforced when databanks are enlarged. Second, we improve the methods for optimizing PB predictions from sequences, revisiting the optimization procedure and exploring different local prediction strategies. Use of a statistical optimization procedure for the sequence-local structure relation improves prediction accuracy by 8% (Q16 = 48.7%). Better recognition of repetitive structures occurs without losing the prediction efficiency of the other local folds. Adding secondary structure prediction improved the accuracy of Q16 by only 1%. An entropy index (Neq), strongly related to the RMSD value of the difference between predicted PBs and true local structures, is proposed to estimate prediction quality. The Neq is linearly correlated with the Q16 prediction rate distributions, computed for a large set of proteins. An "expected" prediction rate QE16 is deduced with a mean error of 5%.     

Temporary secondary structures in tau,an intrinsically disordered protein

The tau protein belongs to the category of intrinsically disordered proteins, which in their native state do not have an average stable structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilising the microtubules in the axons of the neurons. On the other hand, the same tau is involved in the development of Alzheimer disease, when it aggregates in paired helical filaments forming fibrils, which form insoluble tangles. The beginning of the pathological aggregation of tau has been attributed to a local transition of protein portions from random coil to a β-sheet. These structures would very likely be transient; therefore, we performed a molecular dynamics simulation of tau to gather information on the existence of segments of tau endowed with a secondary structure. We combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics a set of most probable conformations of tau. The analysis of these conformations highlights the presence of transient secondary structures such as turns, β-bridges, β-sheets and α-helices. It also shows that a large segment of the N-terminal region is found near the repeats domain in a globular-like shape.     

βVI Turns in peptides and proteins: A model peptide mimicry

To investigate the role of proline in defining β turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L - and D -proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L -proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D -proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala-), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo(-Pro-Ala-Ala-Ala-Ala--), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated. © 1993 Wiley-Liss, Inc.     

Protein local conformations arise from a mixture of Gaussian distributions

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures,which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF's corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end,we have analyzed the correlation between sequence features and the conformational classes.     

Computationally guided identification of Akt-3, a serine/threonine kinase inhibitors: Insights from homology modelling,structure-based screening,molecular dynamics and quantum mechanical calculations

Abstract

Chemical entities targeting kinase signalling pathways serve as a potential strategy to combat malignancies. Protein Kinase B or Akt is a validated target for various malignancies and Akt3 remains the least explored isoform among all its isoforms. Initially, homology modelling technique was used for generating protein structure and further validation was performed using molecular dynamics simulation and Ramachandran plot. The validated protein structure was then subjected for active site analysis which led to identification of active site residues based on metrics provided by site score. The important residues in binding site were identified as Thr81, Asp271 and Asp289 for binding energetics and inhibition. Subsequently, virtual screening methodologies were used for identification of novel hits for inhibition of Protein Kinase B or Akt3. This led to the identification of two hits, i.e. thiophene derivative and thieno-pyridine derivative which were selected on the basis of their binding affinity and drug likeliness. These identified hits were subjected for molecular dynamics simulations, quantum mechanical and synthetic accessibility studies. The role of crucial residues in binding site stood validated as suggested by molecular dynamics simulations studies.

Communicated by Ramaswamy H. Sarma     

Multishelled Gold Nanowires

Abstract

The current miniaturization of electronic devices raises many questions about the properties of various materials at nanometre-scales. Recent molecular dynamics computer simulations have shown that small finite nanowires of gold exist as multishelled structures of lasting stability. These classical simulations are based on a well-tested embedded atom potential. Molecular dynamics simulation studies of metallic nanowires should help in developing methods for their fabrication, such as electron-beam litography and scanning tunneling microscopy.     

Scaling behavior and structure of denatured proteins

An ensemble of random-coil conformations with no persistent structures has long been accepted as the classical model of denatured proteins due to its consistency with the experimentally determined scaling of protein sizes. However, recent NMR spectroscopy studies on proteins at high chemical denaturant concentrations suggest the presence of significant amounts of native-like structures, in contrast to the classical random-coil picture. To reconcile these seemingly controversial observations, we examine thermally denatured states of experimentally characterized proteins by using molecular dynamics simulations. For all studied proteins, we find that denatured states indeed have strong local conformational bias toward native states while a random-coil power law scaling of protein sizes is preserved. In addition, we explain why experimentally determined size of the protein creatine kinase does not follow general scaling. In simulations, we observe that this protein exhibits a stable intermediate state, the size of which is consistent with the reported experimental observation.     

Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Abstract

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1?PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. Abbreviations IDPs intrinsically disordered proteins

REMD replica exchange molecular dynamics

MSM Markov state model

TAD transactivation domain

PH pleckstrin homology

PRR proline-rich region

DBD DNA-binding domain

TET Tetramerization domain

REG regulatory domain

MD molecular dynamics

PME particle-mesh Ewald

TICA time-lagged independent component analysis

CK Chapman–Kolmogorov

GMRQ generalized matrix Rayleigh quotient

SARW self-avoiding random walk

KID kinase-inducible domain

MFPT mean first passage time

DSSP definition of secondary structure of proteins

RMSD root mean square deviation

R_g radius of gyration

R_ee end to end distance

Communicated by Ramaswamy H. Sarma