Holzhütter H G, Frömmel C, Kloetzel P M
Institute of Biochemistry, Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D, 10117, Germany.
J Mol Biol. 1999 Mar 5;286(4):1251-65. doi: 10.1006/jmbi.1998.2530.
Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93 %. The CDAAM is composed of two to four "anchor" positions preferentially located between P5 and P5' around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.
迄今为止，人们对20S蛋白酶体控制肽键选择性切割的机制了解甚少。肽键切割最终可能发生在任一氨基酸残基的羧基位点这一观察结果排除了仅由P1残基简单控制切割偏好的可能性。在此，我们依据这样的基本原理：在可切割肽键周围存在特定的切割决定氨基酸基序（CDAAMs）是实现易于切割的底物构象所必需的。我们基于将给定肽键的切割概率与侧翼氨基酸残基的某些选定侧链性质相关联的经验回归函数，对这些假定的基序进行了探索性搜索。通过将切割概率拟合到一组七种不同肽底物上收集到的关于肽键切割的（二元）实验观察结果，这些底物具有已知的蛋白水解降解产物模式，从而确定相对于可切割键的切割决定残基的序列位置及其最佳侧链性质。在该分析中，假设在P1位置含有相同残基的所有肽键都由蛋白酶体的相同活性位点切割，因此受相同的CDAAMs控制。我们得出了一组最终的十个不同的CDAAMs，它们解释了一到五组不同肽键的切割情况，总体预测正确率为93%。CDAAM由两到四个“锚定”位置组成，这些位置优先位于可切割键周围的P5和P5'之间。这意味着切割位点的使用存在长度限制，这可能会显著抑制较短片段的切除，从而部分解释了观察到的中等大小切割产物占优势的现象。
