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 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.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Amino Acid Substitution, Binding Sites, DNA, DNA-Binding Proteins, Fungal Proteins, Humans, Ligands, Membrane Glycoproteins, Models, Biological, Molecular Sequence Data, Mutation, Nuclear Proteins, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, Protozoan Proteins, Recombinant Fusion Proteins, Repressor Proteins, Saccharomyces cerevisiae Proteins, Substrate Specificity, Transcription Factors, Zinc Fingers
Amino Acid Motifs, Amino Acid Sequence, Amino Acid Substitution, Binding Sites, DNA, DNA-Binding Proteins, Fungal Proteins, Humans, Ligands, Membrane Glycoproteins, Models, Biological, Molecular Sequence Data, Mutation, Nuclear Proteins, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, Protozoan Proteins, Recombinant Fusion Proteins, Repressor Proteins, Saccharomyces cerevisiae Proteins, Substrate Specificity, Transcription Factors, Zinc Fingers
J. Mol. Biol.
Date: Feb. 04, 2000
PubMed ID: 10653693
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