RESUMO
We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.
Assuntos
Química Farmacêutica/métodos , Técnicas de Química Combinatória/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinases , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Ligantes , Espectrometria de Massas/métodos , Modelos Químicos , Estrutura Molecular , Purinas/química , Relação Estrutura-AtividadeRESUMO
The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.
Assuntos
Proteínas de Ligação ao Cálcio/química , Membrana Celular/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/química , Sequência de Aminoácidos/fisiologia , Animais , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Cristalografia por Raios X , Humanos , Proteínas Interatuantes com Canais de Kv , Potenciais da Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida/genética , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Subunidades Proteicas , Canais de Potássio ShalRESUMO
MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.
