Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.
Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.
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泛素依赖性降解底物的晶体结构：溶菌酶的三二硫键形式。
DOI：
10.1073/pnas.90.9.4136
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发表时间：
1993
影响因子：
11.1
通讯作者：
Cohen,RE
中科院分区：
文献类型：
--
作者：
Hill,CP;Johnston,NL;Cohen,RE
泛素的共价连接标志着蛋白质水解的底物，但识别泛素化靶点（如鸡蛋白色溶菌酶）的功能知之甚少。溶菌酶的识别首先需要还原Cys-6 Cys-127，其四个天然二硫键之一，Cys-6，Cys-127-羧甲基化（6，127-rcm）溶菌酶可以模拟这种三硫键中间体。已知6，127-rcm形式的溶菌酶在溶液中保留基本上天然的构象，并且我们证明了这种折叠结构被识别用于泛素化。由于天然溶菌酶不是底物，天然和三个二硫键结构之间的差异必须包括负责选择性泛素化的功能。本文报道的6，127-rcm-溶菌酶的1.9-A分辨率晶体结构提供了这种泛素依赖性降解底物的视图。晶体中有两种6，127-rcm-溶菌酶的构象异构体。这些独特的不同，从天然溶菌酶的晶体形式的C-末端残基的位移。这些结构表明，在三-二硫化物溶菌酶的C末端的局部解折叠允许E3 α（泛素-蛋白连接酶）和E2（泛素-载体蛋白）的复合物结合到包括Lys-1和推定的泛素化位点Lys-13的表面。由此我们推断E3 α E2复合物上的N-末端和内部底物识别位点相隔约20埃。
Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3 alpha (ubiquitin-protein ligase) and E2 (ubiquitin-carrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3 alpha.E2 complex are separated by approximately 20 A.