RESUMO
We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.
Assuntos
Fosfopeptídeos/química , Proteínas/química , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Sequência Consenso , Sequência Conservada , Cristalografia por Raios X , Drosophila , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfopeptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas/metabolismo , Saccharomyces cerevisiae , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM = 0.5 M-1 s-1). These results imply that contacts between Factor D and the C3(H2O)B complex, outside the vicinity of the cleavage site in Factor B, generate >/=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.