RESUMO
Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.
Assuntos
Simulação de Dinâmica Molecular , RNA Polimerase II/química , RNA Polimerase II/genética , Saccharomyces cerevisiae/enzimologia , Thermus thermophilus/enzimologia , Sítios de Ligação , Catálise , Domínio Catalítico , Modelos Moleculares , Conformação Molecular , Mutação/genética , Conformação Proteica , Estrutura Secundária de Proteína , RNA Polimerase II/metabolismoRESUMO
MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFalpha (tumour necrosis factor alpha). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the 'MLK3 protease', reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Western Blotting , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Mutagênese Sítio-Dirigida , Oligopeptídeos/farmacologia , Ésteres de Forbol/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
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 ( approximately 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.
Assuntos
Ácido Aspártico/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , HIV-1/enzimologia , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Farmacorresistência Viral Múltipla , Farmacorresistência Viral , Protease de HIV/genética , Protease de HIV/metabolismo , HIV-1/genética , Humanos , Mutação , Análise de Componente Principal , Conformação Proteica , Solventes/química , Relação Estrutura-Atividade , Água/químicaRESUMO
BACKGROUND: During 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. RESULTS: All 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. CONCLUSIONS: Switching 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.
RESUMO
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.
Assuntos
Ciclo-Oxigenase 1/fisiologia , Peróxido de Hidrogênio/metabolismo , Peroxidases/metabolismo , Sequência de Aminoácidos , Animais , Ciclo-Oxigenase 1/metabolismo , Dimerização , Ativação Enzimática , Humanos , Camundongos , Conformação Molecular , Dados de Sequência Molecular , Consumo de Oxigênio , Homologia de Sequência de Aminoácidos , Ovinos , Especificidade por SubstratoRESUMO
Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane DeltamuH(+) used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a(3) hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. T., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748-5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg(2+), and from E286 to the heme a(3) D-propionate and the binuclear center. The R481K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices III and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI.
Assuntos
Arginina/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Lisina/genética , Mutagênese Sítio-Dirigida , Bombas de Próton/química , Rhodobacter sphaeroides/enzimologia , Água/química , Simulação por Computador , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ácido Glutâmico/química , Heme/análogos & derivados , Heme/química , Heme/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Propionatos/química , Estrutura Secundária de Proteína , Bombas de Próton/metabolismo , Rhodobacter sphaeroides/genética , Termodinâmica , Água/metabolismoRESUMO
Various tyrosyl radicals generated by reaction of both native and indomethacin-inhibited ovine prostaglandin H synthase-1 with ethyl hydrogen peroxide were examined by using high-field/high-frequency EPR spectroscopy. The spectra for the initially formed tyrosyl radical commonly referred to as the "wide-doublet" species and the subsequent "wide-singlet" species as well as the indomethacin-inhibited "narrow-singlet" species were recorded at several frequencies and analyzed. For all three species, the g-values were distributed. In the case of the wide doublet, the high-field EPR spectra indicated that dominant hyperfine coupling was likely to be also distributed. The g(x)-values for all three radicals were found to be consistent with a hydrogen-bonded tyrosyl radical. In the case of the wide-doublet species, this finding is consistent with the known position of the radical and the crystallographic structure and is in contradiction with recent ENDOR measurements. The high-field EPR observations are consistent with the model in which the tyrosyl phenyl ring rotates with respect to both the protein backbone and the putative hydrogen bond donor during evolution from the wide-doublet to the wide-singlet species. The high-field spectra also indicated that the g-values of two types of narrow-singlet species, self-inactivated and indomethacin-inhibited, were likely to be different, raising the possibility that the site of the radical is different or that the binding of the inhibitor perturbs the electrostatic environment of the radical. The 130 GHz pulsed EPR experiments performed on the wide-doublet species indicated that the possible interaction between the radical and the oxoferryl heme species was very weak.
Assuntos
Isoenzimas/química , Prostaglandina-Endoperóxido Sintases/química , Animais , Domínio Catalítico , Ciclo-Oxigenase 1 , Inibidores de Ciclo-Oxigenase/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Ligação de Hidrogênio , Técnicas In Vitro , Indometacina/farmacologia , Modelos Moleculares , Estrutura Molecular , Ovinos , Eletricidade Estática , Tirosina/químicaRESUMO
Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.