Molecular structure of the ParM polymer and the mechanism leading to its nucleotide-driven dynamic instability

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the right-handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra- and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.     

A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities

The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2β, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain–RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain–RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the ‘compensatory’ mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins.     

Allosteric switching by mutually exclusive folding of protein domains

Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.     

Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations

Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the ‘CN network’ hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the ‘CN network’ hypothesis has been revisited and the present study suggested that ‘CN network’ should be accounted in terms of ‘structural contacts’ and ‘structural strengths’ in order to precisely describe order of structural stabilities of TFTs.     

Comparison of the transition states for folding of two Ig-like proteins from different superfamilies

In the "fold approach" proteins with a similar fold but different sequences are compared in order to investigate the relationship between native state structure and folding behaviour. Here we compare the properties of the transition states for folding of TI I27, the 27th immunoglobulin domain from human cardiac titin, and that of TNfn3, the third fibronectin type III domain from human tenascin. Experimental phi-values were used as restraints in molecular dynamics simulations to determine the structures that make up the transition state ensembles (TSEs) for folding of the two proteins. The restrained simulations that we present allow a detailed structural comparison of the two TSEs to be made. Further calculations show explicitly that for both proteins the formation of the interactions involving the residues in the folding nucleus is sufficient for the establishment of the topology of the Ig-like fold. We found that, although the folding nuclei of the two proteins are similar, the packing of the folding nucleus of TI I27 is much tighter than that of TNfn3, reflecting the higher experimental phi-values and beta(T) (Tanford Beta) of TI I27. These results suggest that the folding nucleus can be significantly deformed to accommodate extensive sequence variation while conserving the same folding mechanism.     

A study of comparative modelling,simulation and molecular dynamics of CXCR3 receptor with lipid bilayer

The G-coupled receptors seen on the cell surface are composites with a lipid bilayer. The chemokines are kind of G-coupled receptor which majorly involved in the activation and downstream signalling of the cell. In general, many G-coupled receptors lack their 3D structures which become a hurdle in the drug designing process. In this study, comparative modelling of the CXCR3 receptor was carried out, structure evaluation was done using various tools and softwares. Additionally, molecular dynamics and docking were performed to prove the structural quality and architecture. Interestingly, the studies like toggle switch mechanism, lipid dynamics, virtual screening were carried out to find the potent antagonist for the CXCR3 receptor. During virtual screening 14,303 similar molecules were retrieved among them only four compounds have an ability to interact with a crucial amino acid residue of an antagonist. Hence, these screened compounds can serve as a drug candidate for a CXCR3 receptor, but further in vitro and in vivo studies are ought to do to prove its same efficacy.     

Molecular modeling of pathogenesis-related proteins of family 5

The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of α-amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for earch protein.     

Investigations of Takeout proteins’ ligand binding and release mechanism using molecular dynamics simulation

Takeout (To) proteins exist in a diverse range of insect species. They are involved in many important processes of insect physiology and behaviors. As the ligand carriers, To proteins can transport the small molecule to the target tissues. However, ligand release mechanism of To proteins is unclear so far. In this contribution, the process and pathway of the ligand binding and release are revealed by conventional molecular dynamics simulation, steered molecular dynamics simulation and umbrella sampling methods. Our results show that the α4-side of the protein is the unique gate for the ligand binding and release. The structural analysis confirms that the internal cavity of the protein has high rigidity, which is in accordance with the recent experimental results. By using the potential of mean force calculations in combination with residue cross correlation calculation, we concluded that the binding between the ligand and To proteins is a process of conformational selection. Furthermore, the conformational changes of To proteins and the hydrophobic interactions both are the key factors for ligand binding and release.     

Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes

We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three-state unfolding mechanism with a monomeric intermediate. On the other hand, 3'-azido-3'-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKMT: We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D-structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKMT: This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species-specific antimicrobials.     

Characterization of the hinges of the effector loop in the reaction pathway of the activation of ras-proteins. Kinetics of binding of beryllium trifluoride to V29G and I36G mutants of Ha-ras-p21.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434-451). The process can be studied experimentally by analyzing the binding of BeF3- to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138-23143). Two mutants, V29G and 136G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.     

Analysis of peptides and proteins in their binding to GroEL

The GroEL–GroES is an essential molecular chaperon system that assists protein folding in cell. Binding of various substrate proteins to GroEL is one of the key aspects in GroEL‐assisted protein folding. Small peptides may mimic segments of the substrate proteins in contact with GroEL and allow detailed structural analysis of the interactions. A model peptide SBP has been shown to bind to a region in GroEL that is important for binding of substrate proteins. Here, we investigated whether the observed GroEL–SBP interaction represented those of GroEL–substrate proteins, and whether SBP was able to mimic various aspects of substrate proteins in GroE‐assisted protein folding cycle. We found that SBP competed with substrate proteins, including α‐lactalbumin, rhodanese, and malate dehydrogenase, in binding to GroEL. SBP stimulated GroEL ATP hydrolysis rate in a manner similar to that of α‐lactalbumin. SBP did not prevent GroES from binding to GroEL, and GroES association reduced the ATPase rates of GroEL/SBP and GroEL/α‐lactalbumin to a comparable extent. Binding of both SBP and α‐lactalbumin to apo GroEL was dominated by hydrophobic interaction. Interestingly, association of α‐lactalbumin to GroEL/GroES was thermodynamically distinct from that to GroEL with reduced affinity and decreased contribution from hydrophobic interaction. However, SBP did not display such differential binding behaviors to apo GroEL and GroEL/GroES, likely due to the lack of a contiguous polypeptide chain that links all of the bound peptide fragments. Nevertheless, studies using peptides provide valuable information on the nature of GroEL–substrate protein interaction, which is central to understand the mechanism of GroEL‐assisted protein folding. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Prediction of protein conformational freedom from distance constraints

A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits. These limits depend on distances measured in experimental structures and the strength of the interatomic interaction. Structural differences between generated structures are similar to those obtained from experiment and from MD simulation. Although detailed aspects of dynamical mechanisms are not covered and the extent of variations are only estimated in a relative sense, applications to an IgG-binding domain, an SH3 binding domain, HPr, calmodulin, and lysozyme are presented which illustrate the use of the method as a fast and simple way to predict structural variability in proteins. The method may be used to support the design of mutants, when structural fluctuations for a large number of mutants are to be screened. The results suggest that motional freedom in proteins is ruled largely by a set of simple geometric constraints. Proteins 29:240–251, 1997. © 1997 Wiley-Liss, Inc.     

DNA fine structure and dynamics in crystals and in solution: the impact of BI/BII backbone conformations

Sugar-phosphate backbone conformations are an important structural element for a complete understanding of specific recognition in nucleic acid-protein interactions. They can be involved both in early stages of target discrimination and in structural adaptation upon binding. In the first part of this study, we have analyzed high-resolution structures of double-stranded B-DNA either isolated or bound to proteins, and explored the impact of both the standard BI and the unusual BII phosphate backbone conformations on neighboring sugar puckers and on selected helical parameters. Correlations are found to be similar for free and bound DNA, and in both categories, the possible facing backbone conformations (BI.BI, BI.BII, and BII.BII) define well-characterized substates in the B-DNA conformational space. Notably, BII.BII steps are characterized by specific, and sequence-independent, structural effects involving reduced standard deviations for almost all conformational parameters. In the second part of this work, we analyze four 10 ns molecular dynamics simulations in explicit solvent on the DNA targets of NF-kappaB and bovine papillomavirus E2 proteins, highlighting the multiplicity of backbone dynamical behavior. These results show sequence effects on the percentages of BI and BII conformers, the preferential state of facing backbones, the occurrence of coupled transitions. The backbone states can consequently be seen as a mechanism for transmitting information from the bases to the phosphate groups and thus for modulating the overall structural properties of the target DNA.     

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.     

Mechanistic events underlying odorant binding protein chemoreception

Odorant binding proteins (OBP's) are small hydrophilic proteins, belonging to the lipocalin family dedicated to bind and transport small hydrophobic ligands. Despite many works, the mechanism of ligand binding, together with the functional role of these proteins remains a topic of debate and little is known at the atomic level. The present work reports a computational study of odorants capture and release by an OBP, using both constrained and unconstrained simulations, giving a glimpse on the molecular mechanism of chemoreception. The residues at the origin of the regulation of the protein door opening are identified and a tyrosine amino-acid together with other nearby residues appear to play a crucial role in allowing this event to occur. The simulations reveal that this tyrosine and the protein's L5 loop are implicated in the ligand contact with the protein and act as an anchoring point for the ligand. The protein structural features required for the ligand entry are highly conserved among many transport proteins, suggesting that this mechanism could somewhat be extended to some members of the larger family of lipocalin.     

DMSO enhanced conformational switch of an interfacial enzyme

Interfacial proteins function in unique heterogeneous solvent environments, such as water–oil interfaces. One important example is microbial lipase, which is activated in an oil‐water emulsion phase and has many important enzymatic functions. A unique aprotic dipolar organic solvent, dimethyl sulfoxide (DMSO), has been shown to increase the activity of lipases, but the mechanism behind this enhancement is still unknown. Here, all‐atom molecular dynamics simulations of lipase in a binary solution were performed to examine the effects of DMSO on the dynamics of the gating mechanism. The amphiphilic α5 region of the lipase was a focal point for the analysis, since the structural ordering of α5 has been shown to be important for gating under other perturbations. Compared to the closed‐gorge ensemble in an aqueous environment, the conformational ensemble shifts towards open‐gorge structures in the presence of DMSO solvents. Increased width of the access channel is particularly prevalent in 45% and 60% DMSO concentrations (w/w). As the amount of DMSO increases, the α5 region of the lipase becomes more α‐helical, as we previously observed in studies that address water–oil interfacial and high pressure activation. We believe that the structural ordering of α5 plays an essential role on gating and lipase activity.     

Similarity of domain organization of proteins in phylogenetically distant organisms as a basis of meiosis conservatism

The cytological mechanism of meiosis is very conservative in all eukaryotes. Some meiosis-specific structural proteins of yeasts, nematode Caenorhabditis elegans, Drosophila, and mammals, which play identical roles in cells during meiosis, do not have homology of the primary structure, but their domain organization and conformation are similar. The enzymes of meiotic recombination in yeasts and plants have similar epitopes. These facts suggest that the similarity of the higher level of organization of the meiosis-specific proteins allows these proteins to form similar subcellular structures and produce similar cytological picture of meiosis and similar functions of these subcellular structures. Finally, this leads to a conservative scheme of meiosis in evolutionally distant eukaryotes.     

Preformed structural elements feature in partner recognition by intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) are devoid of extensive structural order but often display signs of local and limited residual structure. To explain their effective functioning, we reasoned that such residual structure can be crucial in their interactions with their structured partner(s) in a way that preformed structural elements presage their final conformational state. To check this assumption, a database of 24 IUPs with known 3D structures in the bound state has been assembled and the distribution of secondary structure elements and backbone torsion angles have been analysed. The high proportion of residues in coil conformation and with phi, psi angles in the disallowed regions of the Ramachandran map compared to the reference set of globular proteins shows that IUPs are not fully ordered even in their bound form. To probe the effect of partner proteins on IUP folding, inherent conformational preferences of IUP sequences have been assessed by secondary structure predictions using the GOR, ALB and PROF algorithms. The accuracy of predicting secondary structure elements of IUPs is similar to that of their partner proteins and is significantly higher than the corresponding values for random sequences. We propose that strong conformational preferences mark regions in IUPs (mostly helices), which correspond to their final structural state, while regions with weak conformational preferences represent flexible linkers between them. In our interpretation, preformed elements could serve as initial contact points, the binding of which facilitates the reeling of the flexible regions onto the template. This finding implies that IUPs draw a functional advantage from preformed structural elements, as they enable their facile, kinetically and energetically less demanding, interaction with their physiological partner.     

The structural basis of mode of activation and functional diversity: a case study with HtrA family of serine proteases

HtrA (High temperature requirement protease A) proteins that are primarily involved in protein quality control belong to a family of serine proteases conserved from bacteria to humans. HtrAs are oligomeric proteins that share a common trimeric pyramidal architecture where each monomer comprises a serine protease domain and one or two PDZ domains. Although the overall structural integrity is well maintained and they exhibit similar mechanism of activation, subtle conformational changes and structural plasticity especially in the flexible loop regions and domain interfaces lead to differences in their active site conformation and hence in their specificity and functions.