A Three-Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity

A three-site mechanism for agonist/antagonist selective binding to vasopressin receptors

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Conformation and dynamics of human urotensin II and urotensin related peptide in aqueous solution

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, <i>open</i> and <i>folded</i>, with rapidly interchanging subtypes. The <i>open</i> states are characterized by turns of various types centered at K⁸Y⁹ or F⁶W⁷ predominantly with no or only sparsely populated transannular hydrogen bonds. The <i>folded</i> conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F⁶W⁷K⁸ or W⁷K⁸Y⁹. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% <i>open</i>:28% <i>folded</i> conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an <i>open:folded</i> equilibrium shifted further toward <i>open</i> conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR

Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.Z.F. and A.F.M.H. were supported by Sir William Dunn School EPA PhD studentships and also a Clarendon Scholarship and a Santander Graduate Award to A.F.M.H; A.C., A.W., M.A.C., P.T.C., and J.W.R. were supported by a Wellcome Trust Senior Investigator Award (104575); S.J. and S.M.L. were supported by a Wellcome Trust Senior Investigator Award (100298); A.W. was also partially supported by a Wellcome Trust Strategic Award to the Micron Oxford Advanced Bioimaging Unit (107457)

Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity

Biomolecular simulation is increasingly central to understanding and designing biological molecules and their interactions. Detailed, physics‐based simulation methods are demonstrating rapidly growing impact in areas as diverse as biocatalysis, drug delivery, biomaterials, biotechnology, and drug design. Simulations offer the potential of uniquely detailed, atomic‐level insight into mechanisms, dynamics, and processes, as well as increasingly accurate predictions of molecular properties. Simulations can now be used as computational assays of biological activity, for example, in predictions of drug resistance. Methodological and algorithmic developments, combined with advances in computational hardware, are transforming the scope and range of calculations. Different types of methods are required for different types of problem. Accurate methods and extensive simulations promise quantitative comparison with experiments across biochemistry. Atomistic simulations can now access experimentally relevant timescales for large systems, leading to a fertile interplay of experiment and theory and offering unprecedented opportunities for validating and developing models. Coarse‐grained methods allow studies on larger length‐ and timescales, and theoretical developments are bringing electronic structure calculations into new regimes. Multiscale methods are another key focus for development, combining different levels of theory to increase accuracy, aiming to connect chemical and molecular changes to macroscopic observables. In this review, we outline biomolecular simulation methods and highlight examples of its application to investigate questions in biology. This article is categorized under: Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Free Energy Method

Structural basis of centrosome assembly in flies

Centrosomes are the main microtubule-organising centres in many animal cells. They consist of a pair of centrioles surrounded by a dense mass of protein known as the pericentriolar material. As the cell enters mitosis, this pericentriolar material expands dramatically in a process called centrosome maturation. In flies, the protein Centrosomin (Cnn) assembles into a network that acts as a scaffold for the recruitment of the hundreds of other proteins making up a mature centrosome. Cnn network formation is known to depend on phosphorylation of a central region within Cnn. However, it is unclear how Cnn assembles into a network and how this process is controlled. In this thesis it is shown that Cnn network formation in vitro depends on an interaction between the conserved C-terminus of Cnn and the phospho-regulated central region. The crystal structure of part of this central region is solved, revealing that it forms a dimer of α-helical hairpins that are sterically incompatible with binding of the Cnn C-terminus. The structure is therefore proposed to constitute an “inactive” state. Furthermore, the structure suggests a mechanism wherein phosphorylation acts by disrupting the hairpin fold, thereby exposing the Cnn C-terminus binding site and hence allowing formation of an “assembly-competent” state where the Cnn C-terminus is bound. In addition, the calcium-binding protein Calmodulin is shown to crosslink the Cnn C-terminus in crystallo. However, Calmodulin inhibits network assembly of Cnn constructs in solution, possibly by preventing the assembly-competent state from forming a network. Taken together, this thesis draws on structural and functional data to provide important atomic-level insights into the mechanism of mitotic centrosome assembly in flies.</p

Can simulations and modeling decipher NMR data for conformational equilibria? Arginine–vasopressin

Arginine vasopressin (AVP) has been suggested by molecular-dynamics (MD) simulations to exist as a mixture of conformations in solution. The ¹H and ¹³C NMR chemical shifts of AVP in solution have been calculated for this conformational ensemble of ring conformations (identified from a 23 μs molecular-dynamics simulation). The relative free energies of these conformations were calculated using classical metadynamics simulations in explicit water. Chemical shifts for representative conformations were calculated using density-functional theory. Comparison with experiment and analysis of the results suggests that the ¹H chemical shifts are most useful for assigning equilibrium concentrations of the conformations in this case. ¹³C chemical shifts distinguish less clearly between conformations, and the distances calculated from the nuclear Overhauser effect do not allow the conformations to be assigned clearly. The ¹H chemical shifts can be reproduced with a standard error of less than 0.24 ppm (<2.2 ppm for ¹³C). The combined experimental and theoretical results suggest that AVP exists in an equilibrium of approximately 70% <i>saddlelike</i> and 30% <i>clinched open</i> conformations. Both newly introduced statistical metrics designed to judge the significance of the results and Smith and Goodman’s DP4 probabilities are presented