RESUMO
In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4ß6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1ß1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the ß2-AR*·Gαsß1γ2 complex crystal structure, the Gαi1ß1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1ß1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptor CB2 de Canabinoide/química , Receptor CB2 de Canabinoide/metabolismo , Cromatografia Líquida , Reagentes de Ligações Cruzadas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Receptor CB2 de Canabinoide/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Orotidine 5'-monophosphate decarboxylase (ODCase) is known as one of the most proficient enzymes. The enzyme catalyzes the last reaction step of the de novo pyrimidine biosynthesis, the conversion from orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate. The enzyme is found in all three domains of life, Bacteria, Eukarya and Archaea. Multiple sequence alignment of 750 putative ODCase sequences resulted in five distinct groups. While the universally conserved DxKxxDx motif is present in all the groups, depending on the groups, several characteristic motifs and residues can be identified. Over 200 crystal structures of ODCases have been determined so far. The structures, together with biochemical assays and computational studies, elucidated that ODCase utilized both transition state stabilization and substrate distortion to accelerate the decarboxylation of its natural substrate. Stabilization of the vinyl anion intermediate by a conserved lysine residue at the catalytic site is considered the largest contributing factor to catalysis, while bending of the carboxyl group from the plane of the aromatic pyrimidine ring of OMP accounts for substrate distortion. A number of crystal structures of ODCases complexed with potential drug candidate molecules have also been determined, including with 6-iodo-uridine, a potential antimalarial agent.