Wilson Jeffrey J, Matsushita Osamu, Okabe Akinobu, Sakon Joshua
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
EMBO J. 2003 Apr 15;22(8):1743-52. doi: 10.1093/emboj/cdg172.
The crystal structure of a collagen-binding domain (CBD) with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined at 1.00 A resolution in the absence of calcium (1NQJ) and at 1.65 A resolution in the presence of calcium (1NQD). The mature enzyme is composed of four domains: a metalloprotease domain, a spacing domain and two CBDs. A 12-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the CBD reveals a beta-sheet sandwich fold with the linker adopting an alpha-helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new beta-strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. Furthermore, extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the collagen-binding surface of the protein and propose likely collagen-protein binding models.
对来自溶组织梭菌I类胶原酶的带有N端结构域连接子的胶原结合结构域（CBD）的晶体结构，在无钙条件下以1.00 Å分辨率（1NQJ）以及在有钙条件下以1.65 Å分辨率（1NQD）进行了测定。成熟酶由四个结构域组成：一个金属蛋白酶结构域、一个间隔结构域和两个CBD。在每个CBD的N端发现了一个12个残基长的连接子。在无钙情况下，CBD呈现出β-折叠三明治结构，连接子呈α-螺旋构象。加入钙后，连接子展开并作为一条新的β-链锚定在三明治结构的远端。通过尺寸排阻色谱以及有无钙条件下的动态光散射测量斯托克斯半径和流体动力学半径的变化，证实了连接子在钙结合时的构象变化。此外，对保守表面残基进行广泛诱变并开展胶原结合研究，使我们能够确定该蛋白的胶原结合表面，并提出可能的胶原-蛋白结合模型。
