Rios-Steiner J L, Schenone M, Mochalkin I, Tulinsky A, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
J Mol Biol. 2001 May 11;308(4):705-19. doi: 10.1006/jmbi.2001.4646.
The X-ray crystal structure of a complex of a modified recombinant kringle-2 domain of human plasminogen, K2Pg[C4G/E56D/L72Y] (mK2Pg), containing an upregulated lysine-binding site, bound to a functional 30 residue internal peptide (VEK-30) from an M-type protein of a group A Streptococcus surface protein, has been determined by molecular replacement methods using K4Pg as a model, and refined at 2.7 A resolution to a R-factor of 19.5 %. The X-ray crystal structure shows that VEK-30 exists as a nearly end-to-end alpha-helix in the complex with mK2Pg. The final structure also revealed that Arg17 and His18 of VEK-30 served as cationic loci for Asp54 and Asp56 of the consensus lysine-binding site of mK2Pg, while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2Pg. The hydrophobic ligand-binding pocket in mK2Pg, consisting primarily of Trp60 and Trp70, situated between the positive and negative centers of the lysine-binding site, is utilized in a novel manner in stabilizing the interaction with VEK-30 by forming a cation-pi-electron-mediated association with the positive side-chain of Arg17 of this peptide. Additional lysine-binding sites, as well as exosite electrostatic and hydrogen bonding interactions involving Glu9 and Lys14 of VEK-30, were observed in the structural model. The importance of these interactions were tested in solution by investigating the binding constants of synthetic variants of VEK-30 to mK2Pg, and it was found that, Lys14, Arg17, His18, and Glu20 of VEK-30 were the most critical amino acid binding determinants. With regard to the solution studies, circular dichroism analysis of the titration of VEK-30 with mK2Pg demonstrated that the peptidic alpha-helical structure increased substantially when bound to the kringle module, in agreement with the X-ray results. This investigation is the first to delineate structurally the mode of interaction of the lysine-binding site of a kringle with an internal pseudo-lysine residue of a peptide or protein that functionally interacts with a kringle module, and serves as a paradigm for this important class of interactions.
已通过分子置换法，以K4Pg为模型，确定了人纤溶酶原修饰重组kringle-2结构域K2Pg[C4G/E56D/L72Y]（mK2Pg）与A组链球菌表面蛋白M型蛋白的功能性30个残基内部肽（VEK-30）结合的复合物的X射线晶体结构，并在2.7 Å分辨率下进行精修，R因子为19.5%。X射线晶体结构表明，在与mK2Pg形成的复合物中，VEK-30以近乎端对端的α螺旋形式存在。最终结构还显示，VEK-30的Arg17和His18作为mK2Pg共有赖氨酸结合位点的Asp54和Asp56的阳离子位点，而VEK-30的Glu20与mK2Pg阳离子结合位点的Arg69配位。mK2Pg中的疏水配体结合口袋主要由Trp60和Trp70组成，位于赖氨酸结合位点的正负中心之间，通过与该肽Arg17的正侧链形成阳离子-π电子介导的缔合，以一种新的方式用于稳定与VEK-30的相互作用。在结构模型中还观察到了额外的赖氨酸结合位点，以及涉及VEK-30的Glu9和Lys14的外部位点静电和氢键相互作用。通过研究VEK-30合成变体与mK2Pg的结合常数，在溶液中测试了这些相互作用的重要性，发现VEK-30的Lys14、Arg17、His18和Glu20是最关键的氨基酸结合决定因素。关于溶液研究，用mK2Pg滴定VEK-30的圆二色性分析表明，与kringle模块结合时，肽的α螺旋结构显著增加，这与X射线结果一致。本研究首次在结构上描绘了kringle赖氨酸结合位点与在功能上与kringle模块相互作用的肽或蛋白质的内部假赖氨酸残基的相互作用模式，并作为这类重要相互作用的范例。
