The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins. KRAB-mediated gene silencing requires binding to the corepressor KAP-1. The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface. Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity. We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components. Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain. A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain. The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions. KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies. Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity. Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding. Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain. The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition. These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins.
Copyright 2000 Academic Press.