RESUMO
C1r is the serine protease (SP) that mediates autoactivation of C1, the complex that triggers the classical complement pathway. We have determined the crystal structure of two fragments from the human C1r catalytic domain, each encompassing the second complement control protein (CCP2) module and the SP domain. The wild-type species has an active structure, whereas the S637A mutant is a zymogen. The structures reveal a restricted hinge flexibility of the CCP2-SP interface, and both are characterized by the unique alpha-helical conformation of loop E. The zymogen activation domain exhibits high mobility, and the active structure shows a restricted access to most substrate binding subsites. Further implications relevant to the C1r self-activation process are derived from protein-protein interactions in the crystals.
Assuntos
Ativação do Complemento , Complemento C1r/química , Precursores Enzimáticos/química , Sítios de Ligação , Domínio Catalítico , Complemento C1r/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
NADP-dependent methylene-H(4)MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP(+) as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A resolution. The enzyme is present as a homotrimer. The alpha,beta fold of the monomer is related to that of methylene-H(4)F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H(4)MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H(4)F dehydrogenases are discussed.
Assuntos
Methylobacterium extorquens/enzimologia , NADP/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Methylobacterium extorquens/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ligação Proteica , Conformação Proteica , Pterinas/metabolismo , Alinhamento de SequênciaRESUMO
Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme contains tightly bound the nickel porphinoid F430. The nickel enzyme has been shown to be active only when its prosthetic group is in the Ni(I) reduced state. In this state MCR exhibits the nickel-based EPR signal red1. We report here for the MCR from Methanothermobacter marburgensis that the EPR spectrum of the active enzyme changed upon addition or removal of coenzyme M, methyl coenzyme M and/or coenzyme B. In the presence of methyl-coenzyme M the red1 signal showed a more resolved 14N-superhyperfine splitting than in the presence of coenzyme M indicating a possible axial ligation of the substrate to the Ni(I). In the presence of methyl-coenzyme M and coenzyme B the red1 signal was the same as in the presence of methyl-coenzyme M alone. However, in the presence of coenzyme M and coenzyme B a highly rhombic EPR signal, MCR-red2, was induced, which was found to be light sensitive and appeared to be formed at the expense of the MCR-red1 signal. Upon addition of methyl-coenzyme M, the red2 signal disappeared and the red1 signal increased again. The red2 signal of MCR with 61Ni-labeled cofactor was significantly broadened indicating that the signal is nickel or nickel-ligand based.
