Reconstitution of the KRAB-KAP-1 repressor complex: A model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions

The KRAB domain is a 75 amino acid residue transcriptional repression modul e commonly found in eukaryotic zinc-finger proteins. KRAB-mediated gene sil encing requires binding to the corepressor KAP-1. The KRAB:KAP-1 interactio n requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a wi dely distributed motif, hypothesized to be a protein-protein interface. Lit tle is known about RBCC-mediated ligand binding and the role of the individ ual sub-domains in recognition and specificity. We have addressed these iss ues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction us ing purified components. Our results show that KRAB binding to KAP-1 is dir ect and specific, as the related RBCC domains from TIF1 alpha and MID1 do n ot bind the KRAB domain. A combination of gel filtration, analytical ultrac entrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC m ust oligomerize likely as a homo-trimer in order to bind the KRAB domain. T he RING finger, B2 box, and coiled-coil region are required for oligomeriza tion of KAP-1-RBCC and KRAB binding, as mutations in these domains concomit antly abolished these functions. KRAB domain binding stabilized the homo-ol igomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies. Mutant KAP-1-RBCC molecules hetero-oligomer ize with the wild-type KAP-1, but these complexes were inactive for KRAB bi nding, suggesting a potential dominant negative activity. Substitution of t he coiled-coil region with heterologous dimerization, trimerization, or tet ramerization domains failed to recapitulate KRAB domain binding. Chimeric K AP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil r egions from MIDI also failed to bind the KRAB domain. The KAP-1-RBCC mediat es a highly specific, direct interaction with the KRAB domain, and it appea rs to function as an integrated, possibly cooperative structural unit where in 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 ligan ds for RBCC domain proteins. (C) 2000 Academic Press.