Hoover G J, Menhart N, Martin A, Warder S, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Biochemistry. 1993 Oct 19;32(41):10936-43. doi: 10.1021/bi00092a002.
A series of strategically designed recombinant (r) mutants of the kringle 1 region of human plasminogen ([K1HPg]) have been constructed and the resulting gene products employed to reveal the identities of the residues that contribute to stabilization of the binding of omega-amino acid ligands to this domain. On the basis of determinations of the binding constants of the ligands, 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, to a variety of these mutants, we find that the anionic site of the polypeptide responsible for stabilization of the amino group of the ligands consists of both D54 and D56 and the cationic site of the polypeptide that interacts with the carboxylate group of the ligand is composed solely of R70. The main hydrophobic interactions that stabilize binding of these ligands, likely by interactions with the ligand hydrophobic regions, are principally due to W61, Y63, and Y71. The results obtained are consistent with conclusions that could be made from analysis of the X-ray crystal structure of r-[K1HPg] and from previous studies from this laboratory regarding the binding of ligands of this type to the kringle 2 region of tissue-type plasminogen activator ([K2tPA]). It thus appears as though a common ligand binding site has evolved in different kringles with ligand specificity differences between r-[K2tPA] and r-[K1HPg] perhaps explainable by the different nature of the cationic sites on these polypeptides that are involved in coordination to the ligand carboxylate groups.
已构建了一系列经过策略性设计的人纤溶酶原 kringle 1 区域（[K1HPg]）的重组（r）突变体，并利用所得的基因产物来揭示有助于稳定 ω-氨基酸配体与该结构域结合的残基的身份。基于对配体 6-氨基己酸和反式-4-(氨甲基)环己烷-1-羧酸与多种这些突变体的结合常数的测定，我们发现负责稳定配体氨基的多肽阴离子位点由 D54 和 D56 组成，而与配体羧基相互作用的多肽阳离子位点仅由 R70 组成。稳定这些配体结合的主要疏水相互作用，可能是通过与配体疏水区域的相互作用，主要归因于 W61、Y63 和 Y71。所得结果与从 r-[K1HPg] 的 X 射线晶体结构分析以及本实验室先前关于此类配体与组织型纤溶酶原激活剂的 kringle 2 区域（[K2tPA]）结合的研究中得出的结论一致。因此，似乎在不同的 kringle 中已经进化出了一个共同的配体结合位点，r-[K2tPA] 和 r-[K1HPg] 之间的配体特异性差异可能可以通过这些多肽上参与与配体羧基配位的阳离子位点的不同性质来解释。
