Peng H, Begg G E, Schultz D C, Friedman J R, Jensen D E, Speicher D W, Rauscher F J
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA.
J Mol Biol. 2000 Feb 4;295(5):1139-62. doi: 10.1006/jmbi.1999.3402.
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.
KRAB 结构域是一个由 75 个氨基酸残基组成的转录抑制模块，常见于真核生物锌指蛋白中。KRAB 介导的基因沉默需要与共抑制因子 KAP-1 结合。KRAB 与 KAP-1 的相互作用需要 KAP-1 的 RING-B 框-卷曲螺旋（RBCC）结构域，这是一个广泛分布的基序，被认为是一个蛋白质-蛋白质相互作用界面。关于 RBCC 介导的配体结合以及各个亚结构域在识别和特异性方面的作用，人们了解甚少。我们通过使用纯化的组分重组并表征 KRAB:KAP-1-RBCC 相互作用来解决这些问题。我们的结果表明，KRAB 与 KAP-1 的结合是直接且特异性的，因为来自 TIF1α 和 MID1 的相关 RBCC 结构域不与 KRAB 结构域结合。凝胶过滤、分析超速离心、化学交联、非变性凝胶电泳和定点诱变技术的结合表明，KAP-1-RBCC 必须可能以同三聚体的形式寡聚化才能结合 KRAB 结构域。RING 指结构、B2 框和卷曲螺旋区域是 KAP-1-RBCC 寡聚化和 KRAB 结合所必需的，因为这些结构域中的突变会同时消除这些功能。通过化学交联和速度沉降研究检测到，KRAB 结构域的结合稳定了 KAP-1-RBCC 的同寡聚状态。突变的 KAP-1-RBCC 分子与野生型 KAP-1 异源寡聚化，但这些复合物对 KRAB 结合无活性，表明存在潜在的显性负活性。用异源二聚化、三聚化或四聚化结构域取代卷曲螺旋区域未能重现 KRAB 结构域的结合。含有来自 MID1 的 RING、RING-B 框或卷曲螺旋区域的嵌合 KAP-1-RBCC 蛋白也未能结合 KRAB 结构域。KAP-１-RBCC 介导与 KRAB 结构域的高度特异性直接相互作用，并且它似乎作为一个整合的、可能协同的结构单元发挥作用其中每个亚结构域都有助于寡聚化和/或配体识别。这些观察结果为 RBCC 结构域介导的蛋白质-蛋白质相互作用提供了首要原则，并对鉴定 RBCC 结构域蛋白的新配体具有启示意义。
