Steve A. Seibold

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

BackgroundDuring elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing.ResultsAll atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop.ConclusionsSwitching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.

Prostaglandin Endoperoxide H Synthases PEROXIDASE HYDROPEROXIDE SPECIFICITY AND CYCLOOXYGENASE ACTIVATION

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic “dome” over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

A molecular dynamics study comparing a wild-type with a multiple drug resistant HIV protease: differences in flap and aspartate 25 cavity dimensions.

HIV proteases can develop resistance to therapeutic drugs by mutating specific residues, but still maintain activity with their natural substrates. To gain insight into why mutations confer such resistance, long (∼70 ns) Molecular Dynamics simulations in explicit solvent were performed on a multiple drug resistant (MDR) mutant (with Asn25 in the crystal structure mutated in silico back to the catalytically active Asp25) and a wild type (WT) protease. HIV proteases are homodimers, with characteristic flap tips whose conformations and dynamics are known to be important influences of ligand binding to the aspartates that form the catalytic center. The WT protease undergoes a transition between 25 and 35 ns that is absent in the MDR protease. The origin of this distinction is investigated using principal component analysis, and is related to differences in motion mainly in the flap region of each monomer. Trajectory analysis suggests that the WT transition arises from a concerted motion of the flap tip distances to their catalytic aspartate residues, and the distance between the two flap tips. These distances form a triangle that in the WT expands the active site from an initial (semi‐open) form to an open form, in a correlated manner. In contrast, the MDR protease remains in a more closed configuration, with uncorrelated fluctuations in the distances defining the triangle. This contrasting behavior suggests that the MDR mutant achieves its resistance to drugs by making its active site less accessible to inhibitors. The migration of water to the active site aspartates is monitored. Water molecules move in and out of the active site and individual waters hydrogen bond to both aspartate carboxylate oxygens, with residence times in the ns time regime. Proteins 2007.