Biochemical characterization of human CLN2, related to a fatal neurodegenerative disease : On the basis of the discovery of a novel family of peptidases

我和我的同事在高分辨率下确定了sedolisin的晶体结构，并定义了一个新的家族，丝氨酸羧基肽酶（sedolisins，MEROPS S53家族）。该家族的独特特征如下：（1）枯草杆菌蛋白酶样折叠，（2）由Ser、Glu和Asp组成的催化三联体，（3）在氧阴离子空穴的形成中涉及谷氨酸。CLN 2在溶酶体蛋白降解中起着关键作用，缺乏这种酶会导致致命的神经退行性疾病（Batten病），本项目通过研究CLN 2的结构与功能关系，分析Batten病的生化过程。（1）h残基Ser 280和Glu 77的催化功能该家族的催化机制被假定为利用谷氨酸（Glu 77）作为从充当亲核试剂的丝氨酸（Ser 280）提取质子的一般碱基。CLN 2的S280 A突变体不显示酶活性。此外，CLN 2被Ac-IAF-CHO灭活，Ac-IAF-CHO与CLN 2共价结合。 关于我们 催化丝氨酸残基。E77 A突变体没有显示出任何显著的酶活性（比野生型酶低1/10^4）。结合结构分析结果，确定了氨基酸残基Ser 280和Glu 77为CLN 2的催化残基。（2）CLN_2的亚位点结构为非素侧只有三个亚位点，素侧至少有三个亚位点（S_3-S_3 '）。CLN 2在P_1位上偏向于Phe、Tyr或Leu，表明S_1亚位点被疏水氨基酸残基占据。CLN 2在P_2位的优势氨基酸为Ala、Arg或Asp。结果表明S_2亚位存在静电相互作用。CLN 2偏爱P_3位的Ala，说明S_3亚位点较小。这里描述的结果与CLN 2的结构模型非常一致。（3）CLN 2结构的同源性建模基于与sedolisin的同源性，建立了CLN 2的三维结构模型。阐明了Asp 132的羧基将延伸到活性位点裂缝中，并作为底物N-末端的锚。（4）CLN 2的晶体结构目前正在对含有和不含有新的基于三肽的抑制剂的CLN 2复合物进行三维结构分析。少

My colleagues and I determined the crystal structure of sedolisin at high resolution and defined a novel family, serine-carboxyl peptidases (sedolisins, MEROPS S53 family). Unique features of this family are as follows ; (1)subtilisin-like fold, (2)catalytic triad consisting of Ser, Glu, and Asp, (3)involvement of asparatate in the formation of an oxyanion hole. CLN2 plays a crucial role in lysosomal protein degradation and deficiency of this enzyme leads to a fatal neurodegenerative disease (Batten disease).In this -project, structure and function relationship of CLN2 was studied for analyzing biochemical process of Batten disease.(1)Catalytic function of h residues Ser280 and Glu77The catalytic mechanism of this family was postulated to utilize glutamate (Glu77) as a general base abstracting a proton from the serine (Ser280) that acts as a nucleophile. The S280A mutant of CLN2 showed no enzymatic activity. In addition, CLN2 was inactivated by Ac-IAF-CHO, which bind covalently to the … More catalytic serine residue. The E77A mutant did not show any significant enzymatic activity (1/10^4 lower than that of wild-type enzyme). Coupled with the results of structure analysis, the residues Ser280 and Glu77 were identified as the catalytic residues of CLN2.(2)Subsite structure of CLN2CLN2 is composed of only three subsites on the non-prime side and at least three on the prime side (S_3-S_3'). CLN2 favored Phe, Tyr, or Leu at the P_1 position, suggesting that the S_1 subsite is occupied by hydrophobic amino acid residues. CLN2 preferred Ala, Arg, or Asp at the P_2 position. The result suggests that the S_2 subsite has electrostatic interactions. CLN2 prefers Ala at the P_3 position, suggesting that the S_3 subsite is small. The results described here are well consistent with the structure model of CLN2.(3)Homology modeling of the structure of CLN2A three-dimensional model of CLN2 was built based on the homology with sedolisin. It was clarified that the carboxyl group of Asp132 would extend out into the active site cleft and act as an anchor of the N-terminal of the substrate.(4)Crystal structure of CLN2Three-dimensional structure analyses of CLN2 complexes with and without new tripeptide-based inhibitors are currently under way. Less