Leung Isabel, Dekel Ayelet, Shifman Julia M, Sidhu Sachdev S
Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1;
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
Proc Natl Acad Sci U S A. 2016 Aug 2;113(31):8705-10. doi: 10.1073/pnas.1524648113. Epub 2016 Jul 19.
A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.
深入了解泛素（Ub）在泛素蛋白酶体系统中识别酶的分子机制对于理解Ub的生物学功能至关重要。许多Ub复合物的结构已被解析，在大多数情况下，这些结构揭示了Ub分子一个公共面上的一个大的结构表位。然而，由于这些相互作用通常较弱，详细绘制单个Ub侧链对亲和力和特异性的功能贡献一直很困难。在这里，我们利用了与特定Ub特异性蛋白酶（USP）紧密结合的Ub变体（Ubv），并使用噬菌体展示和饱和扫描诱变来全面绘制结构表位内的功能表位。我们发现，与USP2或USP21结合的Ubv包含一个非常相似的核心功能表位，或“热点”，主要由与野生型序列保守的位置组成，但也有一些更喜欢突变序列的位置。Ubv核心功能表位与人类USP家族中保守的残基接触，因此它可能对Ub与许多家族成员之间的相互作用很重要。
