Animal Genome & Bioinformatics Division, National Institute of Animal Science, RDA, Suwon 441-706, Korea.
Hanwoo Experiment Station, National Institute of Animal Science, RDA, PyeongChang 232-950, Korea.
Int J Mol Sci. 2014 May 6;15(5):7897-938. doi: 10.3390/ijms15057897.
The activated mammalian CAPN-structures, the CAPN/CAST complex in particular, have become an invaluable target model using the structure-based virtual screening of drug candidates from the discovery phase to development for over-activated CAPN linked to several diseases, such as post-ischemic injury and cataract formation. The effect of Ca²⁺-binding to the enzyme is thought to include activation, as well as the dissociation, aggregation, and autolysis of small regular subunits. Unfortunately, the Ca²⁺-activated enzyme tends to aggregate when provided as a divalent ion at the high-concentration required for the protease crystallization. This is also makes it very difficult to crystallize the whole-length enzyme itself, as well as the enzyme-inhibitor complex. Several parameters that influence CAPN activity have been investigated to determine its roles in Ca²⁺-modulation, autoproteolysis, phosphorylation, and intracellular distribution and inhibition by its endogenous inhibitor CAST. CAST binds and inhibits CAPN via its CAPN-inhibitor domains (four repeating domains 1-4; CAST1-4) when CAPN is activated by Ca²⁺-binding. An important key to understanding CAPN1 inhibition by CAST is to determine how CAST interacts at the molecular level with CAPN1 to inhibit its protease activity. In this study, a 3D structure model of a CAPN1 bound bovine CAST4 complex was built by comparative modeling based on the only known template structure of a rat CAPN2/CAST4 complex. The complex model suggests certain residues of bovine CAST4, notably, the TIPPKYQ motif sequence, and the structural elements of these residues, which are important for CAPN1 inhibition. In particular, as CAST4 docks near the flexible active site of CAPN1, conformational changes at the interaction site after binding could be directly related to CAST4 inhibitory activity. These functional interfaces can serve as a guide to the site-mutagenesis in research on bovine CAPN1 structure-function relationships for the design of small molecules inhibitors to prevent uncontrolled and unspecific degradation in the proteolysis of key protease substrates.
哺乳动物的激活 CAPN 结构，特别是 CAPN/CAST 复合物，已成为一种非常有价值的靶标模型，可用于从发现阶段到开发阶段对候选药物进行基于结构的虚拟筛选，以治疗与多种疾病相关的过度激活的 CAPN，如缺血后损伤和白内障形成。人们认为 Ca²⁺与酶的结合既包括激活，也包括小规则亚基的解离、聚集和自解。不幸的是，当提供高浓度的二价离子时，Ca²⁺激活的酶往往会聚集，而这种浓度对于蛋白酶结晶是必需的。这也使得结晶全长酶本身以及酶-抑制剂复合物变得非常困难。已经研究了几种影响 CAPN 活性的参数，以确定其在 Ca²⁺调节、自水解、磷酸化以及细胞内分布和其内源性抑制剂 CAST 抑制中的作用。当 CAPN 通过 Ca²⁺结合激活时，CAST 通过其 CAPN 抑制剂结构域（四个重复结构域 1-4；CAST1-4）结合并抑制 CAPN。理解 CAST 对 CAPN1 的抑制作用的一个重要关键是确定 CAST 如何在分子水平上与 CAPN1 相互作用以抑制其蛋白酶活性。在这项研究中，基于仅有的大鼠 CAPN2/CAST4 复合物的已知模板结构，通过比较建模构建了一个 CAPN1 结合牛 CAST4 复合物的 3D 结构模型。该复合物模型表明牛 CAST4 的某些残基，特别是 TIPPKYQ 基序序列以及这些残基的结构元件，对 CAPN1 抑制很重要。特别是，当 CAST4 靠近 CAPN1 的柔性活性位点对接时，结合后相互作用位点的构象变化可能与 CAST4 的抑制活性直接相关。这些功能界面可以作为研究牛 CAPN1 结构-功能关系的定点诱变的指导，以设计小分子抑制剂来防止关键蛋白酶底物的蛋白水解中不受控制和非特异性的降解。
