Chang Y, Mochalkin I, McCance S G, Cheng B, Tulinsky A, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Biochemistry. 1998 Mar 10;37(10):3258-71. doi: 10.1021/bi972284e.
The X-ray crystal structure of the recombinant (r) kringle 5 domain of human plasminogen (K5HPg) has been solved by molecular replacement methods using K1HPg as a model and refined at 1.7 A resolution to an R factor of 16.6%. The asymmetric unit of K5HPg is composed of two molecules related by a noncrystallographic 2-fold rotation axis approximately parallel to the z-direction. The lysine binding site (LBS) is defined by the regions His33-Thr37, Pro54-Val58, Pro61-Tyr64, and Leu71-Tyr74 and is occupied in the apo-form by water molecules. A unique feature of the LBS of apo-K5HPg is the substitution by Leu71 for the basic amino acid, arginine, that in other kringle polypeptides forms the donor cationic center for the carboxylate group of omega-amino acid ligands. While wild-type (wt) r-K5HPg interacted weakly with these types of ligands, replacement by site-directed mutagenesis of Leu71 by arginine led to substantially increased affinity of the ligands for the LBS of K5HPg. As a result, binding of omega-amino acids to this mutant kringle (r-K5HPg[L71R]) was restored to levels displayed by the companion much stronger affinity HPg kringles, K1HPg and K4HPg. Correspondingly, alkylamine binding to r-K5HPg[L71R] was considerably attenuated from that shown by wtr-K5HPg. Thus, employing a rational design strategy based on the crystal structure of K5HPg, successful remodeling of the LBS has been accomplished, and has resulted in the conversion of a weak ligand binding kringle to one that possesses an affinity for omega-amino acids that is similar to K1HPg and K4HPg.
人纤溶酶原重组（r）kringle 5结构域（K5HPg）的X射线晶体结构已通过分子置换法解析，以K1HPg为模型，并在1.7 Å分辨率下精修至R因子为16.6%。K5HPg的不对称单元由两个通过近似平行于z方向的非晶体学2重旋转轴相关的分子组成。赖氨酸结合位点（LBS）由His33 - Thr37、Pro54 - Val58、Pro61 - Tyr64和Leu71 - Tyr74区域定义，在脱辅基形式中被水分子占据。脱辅基K5HPg的LBS的一个独特特征是Leu71取代了碱性氨基酸精氨酸，而在其他kringle多肽中，精氨酸形成了ω-氨基酸配体羧酸盐基团的供体阳离子中心。虽然野生型（wt）r - K5HPg与这些类型的配体相互作用较弱，但通过定点诱变将Leu71替换为精氨酸导致配体对K5HPg的LBS的亲和力大幅增加。结果，ω-氨基酸与这种突变kringle（r - K5HPg[L71R]）的结合恢复到了与之相伴的亲和力更强的HPg kringle，即K1HPg和K4HPg所显示的水平。相应地，烷基胺与r - K5HPg[L71R]的结合相比wt r - K5HPg有显著减弱。因此，基于K5HPg的晶体结构采用合理设计策略，成功实现了LBS的重塑，并导致了一个弱配体结合kringle转变为一个对ω-氨基酸具有与K1HPg和K4HPg相似亲和力的kringle。
