Electrostatic control of proton pumping in cytochrome c oxidase

AbstractAs part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O2 to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway

An Iris-Like Mechanism of Pore Dilation in the CorA Magnesium Transport System

AbstractMagnesium translocation across cell membranes is essential for numerous physiological processes. Three recently reported crystal structures of the CorA magnesium transport system revealed a surprising architecture, with a bundle of giant α-helices forming a 60-Å-long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. The presence of divalent cations in putative intracellular regulation sites suggests that these structures correspond to the closed conformation of CorA. To examine the nature of the conduction pathway, we performed 110-ns molecular-dynamics simulations of two of these structures in a lipid bilayer with and without regulatory ions. The results show that a 15-Å-long hydrophobic constriction straddling the membrane-cytosol interface constitutes a steric bottleneck whose location coincides with an electrostatic barrier opposing cation translocation. In one of the simulations, structural relaxation after the removal of regulatory ions led to concerted changes in the tilt of the pore helices, resulting in iris-like dilation and spontaneous hydration of the hydrophobic neck. This simple and robust mechanism is consistent with the regulation of pore opening by intracellular magnesium concentration, and explains the unusual architecture of CorA

Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu

Conformational Determinants of Phosphotyrosine Peptides Complexed with the Src SH2 Domain

The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA) to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the “two-pronged plug two-hole socket” model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an α-helix. In contrast, a β-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors

Improving Internal Peptide Dynamics in the Coarse-Grained MARTINI Model: Toward Large-Scale Simulations of Amyloid- and Elastin-like Peptides

We present an extension of the coarse-grained MARTINI model for proteins and apply this extension to amyloid- and elastin-like peptides. Atomistic simulations of tetrapeptides, octapeptides, and longer peptides in solution are used as a reference to parametrize a set of pseudodihedral potentials that describe the internal flexibility of MARTINI peptides. We assess the performance of the resulting model in reproducing various structural properties computed from atomistic trajectories of peptides in water. The addition of new dihedral angle potentials improves agreement with the contact maps computed from atomistic simulations significantly. We also address the question of which parameters derived from atomistic trajectories are transferable between different lengths of peptides. The modified coarse-grained model shows reasonable transferability of parameters for the amyloid- and elastin-like peptides. In addition, the improved coarse-grained model is also applied to investigate the self-assembly of β-sheet forming peptides on the microsecond time scale. The octapeptides SNNFGAIL and (GV)4 are used to examine peptide aggregation in different environments, in water, and at the water–octane interface. At the interface, peptide adsorption occurs rapidly, and peptides spontaneously aggregate in favor of stretched conformers resembling β-strands

On the molecular basis of uracil recognition in DNA: comparative study of T-A versus U-A structure, dynamics and open base pair kinetics

Uracil (U) can be found in DNA as a mismatch paired either to adenine (A) or to guanine (G). Removal of U from DNA is performed by a class of enzymes known as uracil–DNA–glycosylases (UDG). Recent studies suggest that recognition of U–A and U–G mismatches by UDG takes place via an extra-helical mechanism. In this work, we use molecular dynamics simulations to analyze the structure, dynamics and open base pair kinetics of U–A base pairs relative to their natural T–A counterpart in 12 dodecamers. Our results show that the presence of U does not alter the local conformation of B-DNA. Breathing dynamics and base pair closing kinetics are only weakly dependent on the presence of U versus T, with open T–A and U–A pairs lifetimes in the nanosecond timescale. Additionally, we observed spontaneous base flipping in U–A pairs. We analyze the structure and dynamics for this event and compare the results to available crystallographic data of open base pair conformations. Our results are in agreement with both structural and kinetic data derived from NMR imino proton exchange measurements, providing the first detailed description at the molecular level of elusive events such as spontaneous base pair opening and flipping in mismatched U–A sequences in DNA. Based on these results, we propose that base pair flipping can occur spontaneously at room temperature via a 3-step mechanism with an open base pair intermediate. Implications for the molecular basis of U recognition by UDG are discussed