Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein
Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein
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DOI：
10.1006/jmbi.2001.4646
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发表时间：
2001-05-11
影响因子：
5.6
通讯作者：
Castellino, FJ
中科院分区：
文献类型：
--
作者：
Rios-Steiner, JL;Schenone, M;Castellino, FJ
以K4（Pg）为模型，用分子置换法测定了人纤溶酶原修饰的重组kringle-2结构域K2(Pg)[C(4)G/(ED)-D-56/L(72)y] （MK2(Pg)）与a群链球菌表面蛋白m型蛋白的功能性30残基内肽（VEK-30）结合的赖氨酸结合位点上调的配合物的x射线晶体结构，并在2.7埃分辨率下精化到x因子为19.5%。x射线晶体结构表明，在与mK2（Pg）的配合物中，VEK-30以近端对端的α -螺旋结构存在。最终的结构还表明，VEK-30的Arg17和His18是mK2的赖氨酸结合位点的Asp54和Asp56的阳离子位点（Pg），而VEK-30的Glu20与mK2的阳离子结合位点Arg69的阳离子位点（Pg）。mK2（Pg）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，通过与该肽的Arg17的正侧链形成阳离子-pi -电子介导的结合，以一种新的方式用于稳定与VEK-30的相互作用。在结构模型中观察到额外的赖氨酸结合位点，以及涉及VEK-30的Glu9和Lys14的外源静电和氢键相互作用。通过研究VEK-30合成变异体与mK2（Pg）的结合常数，在溶液中验证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子。在溶液研究方面，用mK2（Pg）滴定VEK-30的圆二色性分析表明，当与kringle模块结合时，肽a-螺旋结构显著增加，与x射线结果一致。这项研究首次从结构上描述了kringle的赖氨酸结合位点与与kringle模块功能相互作用的肽或蛋白质的内部伪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。(C) 2001学术出版社。
The X-ray crystal structure of a complex of a modified recombinant kringle-2 domain of human plasminogen, K2(Pg)[C(4)G/(ED)-D-56/L(72)y] (MK2(Pg)), 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 K4(Pg) as a model, and refined at 2.7 Angstrom resolution to a X-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 mK2(Pg). 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 mK2(Pg), while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2(Pg). The hydrophobic ligand-binding pocket in mK2(Pg), 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 mK2(Pg), 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 mK2(Pg) demonstrated that the peptidic a-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. (C) 2001 Academic Press.