Gakhar Lokesh, Bassuk Alexander G, Velez Gabriel, Khan Saif, Yang Jing, Tsang Stephen H, Mahajan Vinit B
Department of Biochemistry, University of Iowa, Iowa City, IA, USA; Protein Crystallography Facility, University of Iowa, Iowa City, IA, USA.
Department of Pediatrics, University of Iowa, Iowa City, IA, USA; Omics Lab, University of Iowa, Iowa City, IA, USA.
J Struct Biol. 2016 Dec;196(3):309-318. doi: 10.1016/j.jsb.2016.07.017. Epub 2016 Jul 27.
Calpain-5 is a calcium-activated protease expressed in the retina. Mutations in calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM#193235). The structure of calpain-5 has not been determined, thus hindering the investigation of its proteolytic targets and pathological role in ADNIV. Herein, we report models of the proteolytic core of calpain-5 (mini-calpain-5) containing two globular domains (termed DIIa-IIb) connected by a short, flexible linker, consistent with small-angle X-ray scattering (SAXS) data. Structural modeling in the absence of calcium suggests that mini-calpain-5 adopts a more open conformation when compared to previously determined structures of other calpain cores. This open conformation, achieved by a rotation of DIIa and DIIb with respect to each other, prevents formation of the active site and constrains the enzyme in an inactivated form. The relative domain rotation of 60-100° we found for mini-calpain-5 (a non-classical calpain) is significantly greater than the largest rotation previously observed for a classical calpain (i.e., 55.0° for mini-calpain-9). Together with our prediction that, in the full-length form, a long loop in DIIb (loop C1), a few residues downstream of the inter-domain linker, likely interacts with the shorter, acidic, inactivating loop on domain-III (DIII), these structural insights illuminate the complexity of calpain regulation. Moreover, our studies argue that pursuing higher resolution structural studies are necessary to understand the complex activity regulation prevalent in the calpain family and for the design of specific calpain inhibitors.
钙蛋白酶-5是一种在视网膜中表达的钙激活蛋白酶。钙蛋白酶-5的突变会导致常染色体显性遗传性新生血管性炎性玻璃体视网膜病变（ADNIV，OMIM编号：193235）。钙蛋白酶-5的结构尚未确定，因此阻碍了对其蛋白水解靶点及其在ADNIV中的病理作用的研究。在此，我们报告了钙蛋白酶-5蛋白水解核心（微型钙蛋白酶-5）的模型，该模型包含两个通过短的柔性连接子相连的球状结构域（称为DIIa-IIb），与小角X射线散射（SAXS）数据一致。在没有钙的情况下进行的结构建模表明，与先前确定的其他钙蛋白酶核心结构相比，微型钙蛋白酶-5呈现出更开放的构象。这种开放构象是通过DIIa和DIIb相互旋转实现的，它阻止了活性位点的形成，并将酶限制在失活形式。我们发现微型钙蛋白酶-5（一种非典型钙蛋白酶）的相对结构域旋转角度为60-100°，明显大于先前观察到的典型钙蛋白酶的最大旋转角度（即微型钙蛋白酶-9为55.0°）。结合我们的预测，即全长形式下，DIIb中的一个长环（环C1），在结构域间连接子下游的几个残基，可能与结构域III（DIII）上较短的酸性失活环相互作用，这些结构见解揭示了钙蛋白酶调节的复杂性。此外，我们的研究表明，进行更高分辨率的结构研究对于理解钙蛋白酶家族中普遍存在的复杂活性调节以及设计特异性钙蛋白酶抑制剂是必要的。
