A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex.

DNA telomeric repeats in mammalian cells are transcribed to guanine-rich RNA sequences, which adopt parallel-stranded G-quadruplexes with a propeller-like fold. The successful crystallization and structure analysis of a bimolecular human telomeric RNA G-quadruplex, folded into the same crystalline environment as an equivalent DNA oligonucleotide sequence, is reported here. The structural basis of the increased stability of RNA telomeric quadruplexes over DNA ones and their preference for parallel topologies is described here. Our findings suggest that the 2'-OH hydroxyl groups in the RNA quadruplex play a significant role in redefining hydration structure in the grooves and the hydrogen bonding networks. The preference for specific nucleotides to populate the C3'-endo sugar pucker domain is accommodated by alterations in the phosphate backbone, which leads to greater stability through enhanced hydrogen bonding networks. Molecular dynamics simulations on the DNA and RNA quadruplexes are consistent with these findings. The computations, based on the native crystal structure, provide an explanation for RNA G-quadruplex ligand binding selectivity for a group of naphthalene diimide ligands as compared to the DNA G-quadruplex

Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis

Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles

Crystal structure of the third KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.6 Å resolution

KH (hnRNP K homology) domains, consisting of ∼70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 Å resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5′-AACCCTA-3′) corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a βααββα configuration. The protein–DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein–protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT

Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii

AbstractBackground: The proteins of halophilic archaea require high salt concentrations both for stability and for activity, whereas they denature at low ionic strength. The structural basis for this phenomenon is not yet well understood. The crystal structure of dihydrofolate reductase (DHFR) from Haloferax volcanii (hv-DHFR) reported here provides the third example of a structure of a protein from a halophilic organism. The enzyme is considered moderately halophilic, as it retains activity and secondary structure at monovalent salt concentrations as low as 0.5 M.Results: The crystal structure of hv-DHFR has been determined at 2.6 å resolution and reveals the same overall fold as that of other DHFRs. The structure is in the apo state, with an open conformation of the active-site gully different from the open conformation seen in other DHFR structures. The unique feature of hv-DHFR is a shift of the α helix encompassing residues 46–51 and an accompanied altered conformation of the ensuing loop relative to other DHFRs. Analysis of the charge distribution, amino acid composition, packing and hydrogen-bonding pattern in hv-DHFR and its non-halophilic homologs has been performed.Conclusions: The moderately halophilic behavior of hv-DHFR is consistent with the lack of striking structural features expected to occur in extremely halophilic proteins. The most notable feature of halophilicity is the presence of clusters of non-interacting negatively charged residues. Such clusters are associated with unfavorable electrostatic energy at low salt concentrations, and may account for the instability of hv-DHFR at salt concentrations lower than 0.5 M. With respect to catalysis, the open conformation seen here is indicative of a conformational transition not reported previously. The impact of this conformation on function and/or halophilicity is unknown

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Arrar, Mehrnoosh. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Sycz, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cerutti, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; ArgentinaFil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Paris, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Marti, Marcelo Adrian. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; Argentin

Classical And Quantum Mechanical Simulations Of Condensed Systems And Biomolecules

This work describes the fundamental study of two enzymes of Fe(II)/-KG super family enzymes (TET2 and AlkB) by applying MD and QM/MM approaches, as well as the development of multipolar-polarizable force field (AMOEBA/GEM-DM) for condensed systems (ionic liquids and water). TET2 catalytic activity has been studied extensively to identify the potential source of its substrate preference in three iterative oxidation steps. Our MD results along with some experimental data show that the wild type TET2 active site is shaped to enable higher order oxidation. We showed that the scaffold stablished by Y1902 and T1372 is required for iterative oxidation. The mutation of these residues perturbs the alignment of the substrate in the active site, resulting in “5hmC-stalling” phenotype in some of the mutants. We provided more details on 5hmC to 5fC oxidation mechanism for wild type and one of the “5hmC-stallling” mutants (E mutant). We showed that 5hmC oxidizes to 5fC in the wild type via three steps. The first step is the hydrogen atom abstraction from hydroxyl group of 5hmC, while the second hydrogen is transferred from methylene group of 5hmC through the third transition state as a proton. Our results suggest that the oxidation in E mutant is kinetically unfavorable due to its high barrier energy. Many analyses have been performed to qualitatively describe our results and we believed our results can be used as a guide for other researchers. In addition, two MD approaches (explicit ligand sampling and WHAM) are used to study the oxygen molecule diffusion into the active site of AlkB. Our results showed that there are two possible channels for oxygen diffusion, however, diffusion through one of them is thermodynamically favorable. We also applied multipolar-polarizable force field to describe the oxygen diffusion along the preferred tunnel. We showed that the polarizable force field can describe the behavior of the highly polarizable systems accurately. We also developed a new multipolar-polarizable force field (AMOEBA/GEM-DM) to calculate the properties of imidazolium- and pyrrolidinium- based ionic liquids and water in a range of temperature. Our results agree well with the experimental data. The good agreement between our results and experimental data is because our new parameters provide an accurate description of non-bonded interactions. We fit all the non-bonded parameters against QM. We use the multipoles extracted from fitted electron densities (GEM) and we consider both inter- and intra-molecular polarization. We believe this method can accurately calculate the properties of condensed systems and can be helpful for designing new systems such as electrolytes

Aggregation of gluten proteins - from wheat seed biology to hydrogels : scientific modelling based primarily on Monte-Carlo and HPLC methods

Gluten proteins are intrinsically disordered proteins that form extensive aggregated networks in wheat seeds, where they are stored as a nutrient source for the embryo. A modelling approach involving computational biology with Monte-Carlo algorithms and wet laboratory studies, including HPLC analysis, was applied to unravel the aggregational and hydrogelforming properties of the gluten proteins. Two of the gluten proteins, “αgliadin” and “low molecular weight glutenin subunits” (LMW-GS) were found to have similar size, folding of disordered, rigid and compact structures, elliptical shape and secondary structures of random coils and turns. Both proteins also share an evolutionarily conserved motif resulting in internal disulphide bonds, which were shown to be established through hydrophobic interactions, together with the inherent order of cysteines. In laboratory conditions and simulations, it was found that gliadins formed oligomers by hydrophobic interactions and cross-links by disulphide and lanthionine bonds at peptide sections in the C-terminal part of the protein. At the N-terminal part, the protein formed oligomers by liquid-liquid phase separation, polyproline II structures and β-sheets. Heat and alkaline treatment was shown to favour cross-linking by lanthionine, lysinoalanine and disulphide bonds among gliadins and increase their ability to absorb liquid. Thus the modelling approach successfully characterised the gluten proteins α-gliadin and LMW-GS, the mechanisms by which they form internal and external cross-links, how they merge into oligomers and how to increase their liquid absorption

Deciphering Chemomechanical Couplings in Proteins Using Microsecond-level Molecular Dynamics Simulations

All-atom molecular dynamics (MD) simulations combine the high temporal resolution of experimental methods like smFRET and spatial resolution of methods like x-ray crystallography, to provide a detailed dynamic picture of biomolecular processes. Here, microsecond-level atomistic MD simulations have been used to characterize chemomechanical couplings in human fibroblast growth factor 1 (hFGF1) and the spike proteins of SARS CoV-1 and SARS-CoV-2. hFGF1 is a globular signaling protein that is involved in several physiological processes ranging from cell proliferation to wound healing. Experimental studies have previously described the low proteolytic and thermal stability of hFGF1, in addition to the stabilizing role of heparin. Here, a conformational change in the hFGF1 heparin-binding pocket that occurs only when heparin is absent, is described for the first time. Comparisons with experimental data indicate that this conformational transition is implicated in the low thermal stability of hFGF1. Unique electrostatic interactions that contribute to heparin-mediated stabilization are also described. This work also describes a novel binding affinity estimation approach involving restrained umbrella sampling simulations. The absolute binding affinity for the hFGF1-heparin interaction determined using this approach is in very good agreement with data from isothermal titration calorimetry (ITC) experiments. This binding affinity study revealed that restraining ligand orientation is essential for effective sampling along a protein-ligand distance collective variable.The differential dynamic behavior of the SARS-CoV-1 and CoV-2 spike proteins is also described in this work. Spike protein activation is the first step in the “effective binding” process leading to interaction with the human ACE2 receptor. This study shows that the active form of the CoV-1 spike protein is less stable than that of the CoV-2 spike protein and that the energy barriers associated with activation and inactivation are higher in CoV-2. A “pseudo-inactive” state of the CoV-1 spike protein is described for the first time, wherein the N-terminal domain (NTD) interacts with the receptor-binding domain (RBD). This highlights the potential role of the NTD in spike protein inactivation. The relatively slower kinetics of spike protein activation and inactivation in CoV-2 indicate that it might spend more time bound to the ACE2 receptor than CoV-1, which in turn might provide an explanation for the higher transmissibility of CoV-2