RESUMO
To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg(2+) or Mn(2+). The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide gamma-phosphate and its associated Mg(2+) ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Thermotoga maritima/enzimologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sítios de Ligação , Cátions Bivalentes/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , Histidina Quinase , Ligação de Hidrogênio , Magnésio/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas Quimiotáticas Aceptoras de Metil , Modelos Moleculares , Fosforilação , Inibidores de Proteínas Quinases , Estrutura Terciária de Proteína , SolventesRESUMO
Cobalt(III) Schiff base complexes have been shown to inhibit the replication of the ocular herpes virus. It is well known that these complexes have a high affinity for nitrogenous donors such as histidine residues, and it is possible that they bind to (and inhibit) an enzyme that is crucial to viral replication. In model studies, we have found that [Co(acacen)(NH3)2]+ is an effective irreversible inhibitor of thermolysin at millimolar concentrations; it also inhibits human alpha-thrombin. Axial ligand exchange with an active-site histidine is the proposed mechanism of inhibition. The activity of thermolysin and thrombin can be protected by binding a reversible inhibitor to the active site before addition of the cobalt(III) complex.
