Padmanabhan K, Padmanabhan K P, Ferrara J D, Sadler J E, Tulinsky A
Department of Chemistry, Michigan State University, East Lansing 48824-1322.
J Biol Chem. 1993 Aug 25;268(24):17651-4. doi: 10.2210/pdb1hut/pdb.
The structure of a complex between human alpha-thrombin and a GGTTGGTGTGGTTGG 15-nucleotide consensus sequence has been solved by x-ray crystallography and refined at 2.9-A resolution to an R value of 0.159. As in solution, in the complex the single-stranded DNA folds into a structure with two G-quartets. The DNA is sandwiched between two different positively charged regions of two symmetry-related thrombin molecules in the crystal structure making ionic and hydrophobic interactions. One region is the fibrinogen recognition exosite and the other, the putative heparin binding site. The lack of inhibition of fibrinogen clotting and platelet activation by the DNA 15-mer with the Arg75-->Glu mutant of thrombin is consistent with the several salt bridges of the DNA in the fibrinogen exosite. The association of DNA with the heparin site of a neighboring molecule appears to simply compensate residual charge. Differences in the 15-mer loop conformations between the complex and NMR solution structures can be attributed to conformational changes upon thrombin binding. Although G-quadruplexes are favored in the presence of monovalent cations, there is no evidence of the latter in the thrombin complex.
人α-凝血酶与GGTTGGTGTGGTTGG 15核苷酸共有序列形成的复合物结构已通过X射线晶体学解析，并在2.9埃分辨率下精修至R值为0.159。与在溶液中一样，在复合物中，单链DNA折叠成具有两个G-四联体的结构。在晶体结构中，DNA夹在两个对称相关的凝血酶分子的两个不同带正电区域之间，形成离子和疏水相互作用。一个区域是纤维蛋白原识别外位点，另一个是假定的肝素结合位点。具有凝血酶Arg75→Glu突变体的DNA 15聚体对纤维蛋白原凝血和血小板激活缺乏抑制作用，这与DNA在纤维蛋白原外位点的几个盐桥一致。DNA与相邻分子的肝素位点的结合似乎只是为了补偿剩余电荷。复合物与核磁共振溶液结构之间15聚体环构象的差异可归因于凝血酶结合后的构象变化。尽管在单价阳离子存在下G-四链体更受青睐，但在凝血酶复合物中没有后者存在的证据。
