Le Guezennec Xavier, Vriend Gert, Stunnenberg Hendrik G
Department of Molecular Biology, Centre for Molecular and Biomolecular Informatics, University of Nijmegen, 6500 HB Nijmegen, The Netherlands.
J Biol Chem. 2004 Jun 11;279(24):25823-9. doi: 10.1074/jbc.M313860200. Epub 2004 Mar 26.
The Sin3 co-repressor acts as a protein scaffold to recruit transcription factors via its four highly homologous paired amphipathic helix (PAH) domains. PAH2 has been shown to interact strongly with the Sin3 interacting domain (SID) of the tumor suppressor Mad. This PAH2/Mad complex has been studied extensively by NMR, but the molecular determinants that dictate the specificity of interaction remain to be elucidated. To uncover residues that convey the specificity of interaction between PAH2 and Mad, PAH2 residues contacted by the Mad-SID were introduced into the PAH1 domain of mSin3b and tested for gain-of-interaction in vivo in a yeast two-hybrid setting and further confirmed in a cell-free system. This approach led to the identification of PAH2-Phe-7 as a critical residue. Stabilization of the interaction between PAH1-Phe-7 and the Mad-SID was achieved by introducing Val-14 and Gln-39 into PAH1. Substitution of PAH2 residues contacted by the Mad-SID with their respective residues in PAH1 corroborated and extended the critical role of Phe-7 and the stabilizing role of Val-14 and Gln-39. We conclude that Phe-7 is the critical determinant and provides the molecular specificity for the association between Sin3 and Mad in regulating cell growth and differentiation.
Sin3共抑制因子作为一种蛋白质支架，通过其四个高度同源的双性α-螺旋（PAH）结构域招募转录因子。PAH2已被证明与肿瘤抑制因子Mad的Sin3相互作用结构域（SID）有强烈相互作用。PAH2/Mad复合物已通过核磁共振进行了广泛研究，但决定相互作用特异性的分子决定因素仍有待阐明。为了揭示传递PAH2与Mad之间相互作用特异性的残基，将与Mad-SID接触的PAH2残基引入mSin3b的PAH1结构域，并在酵母双杂交体系中进行体内相互作用增强测试，并在无细胞体系中进一步证实。这种方法导致鉴定出PAH2-Phe-7是一个关键残基。通过将Val-14和Gln-39引入PAH1，实现了PAH1-Phe-7与Mad-SID之间相互作用的稳定。用PAH1中各自的残基取代与Mad-SID接触的PAH2残基，证实并扩展了Phe-7的关键作用以及Val-14和Gln-39的稳定作用。我们得出结论，Phe-7是关键决定因素，为Sin3和Mad在调节细胞生长和分化中的关联提供分子特异性。
