Rauh Daniel, Klebe Gerhard, Stubbs Milton T
Institut für Pharmazeutische Chemie der Philipps, Universität Marburg, Marbacher Weg 6, D35032 Marburg, Germany.
J Mol Biol. 2004 Jan 30;335(5):1325-41. doi: 10.1016/j.jmb.2003.11.041.
In order to design selective, high-affinity ligands to a target protein, it is advantageous to understand the structural determinants for protein-ligand complex formation at the atomic level. In a model system, we have successively mapped the factor Xa binding site onto trypsin, showing that certain mutations influence both protein structure and inhibitor specificity. Our previous studies have shown that introduction of the 172SSFI175 sequence of factor Xa into rat or bovine trypsin results in the destabilisation of the intermediate helix with burial of Phe174 (the down conformation). Surface exposure of the latter residue (the up conformation) is critical for the correct formation of the aromatic box found in factor Xa-ligand complexes. In the present study, we investigate the influence of aromatic residues in position 174. Replacement with the bulky tryptophan (SSWI) shows reduced affinity for benzamidine-based inhibitors (1) and (4), whereas removal of the side-chain (alanine, SSAI) or exchange with a hydrophilic residue (arginine, SSRI) leads to a significant loss in affinity for all inhibitors studied. The variants could be crystallised in the presence of different inhibitors in multiple crystal forms. Structural characterisation of the variants revealed three different conformations of the intermediate helix and 175 loop in SSAI (down, up and super-up), as well as a complete disorder of this region in one crystal form of SSRI, suggesting that the compromised affinity of these variants is related to conformational flexibility. The influence of Glu217, peripheral to the ligand-binding site in factor Xa, was investigated. Introduction of Glu217 into trypsin variants containing the SSFI sequence exhibited enhanced affinity for the factor Xa ligands (2) and (3). The crystal structures of these variants also exhibited the down and super-up conformations, the latter of which could be converted to up upon soaking and binding of inhibitor (2). The improved affinity of the Glu217-containing variants appears to be due to a shift towards the up conformation. Thus, the reduction in affinity caused by conformational variability of the protein target can be partially or wholly offset by compensatory binding to the up conformation. The insights provided by these studies will be helpful in improving our understanding of ligand binding for the drug design process.
为了设计针对目标蛋白的选择性、高亲和力配体，在原子水平上了解蛋白质 - 配体复合物形成的结构决定因素是很有帮助的。在一个模型系统中，我们已相继将因子Xa结合位点映射到胰蛋白酶上，表明某些突变会影响蛋白质结构和抑制剂特异性。我们之前的研究表明，将因子Xa的172SSFI175序列引入大鼠或牛胰蛋白酶会导致中间螺旋不稳定，苯丙氨酸174（向下构象）被掩埋。后一个残基的表面暴露（向上构象）对于因子Xa - 配体复合物中发现的芳香盒的正确形成至关重要。在本研究中，我们研究了174位芳香族残基的影响。用庞大的色氨酸取代（SSWI）显示对基于苯甲脒的抑制剂（1）和（4）的亲和力降低，而去除侧链（丙氨酸，SSAI）或用亲水性残基取代（精氨酸，SSRI）会导致对所有研究的抑制剂的亲和力显著丧失。这些变体可以在存在不同抑制剂的情况下以多种晶体形式结晶。变体的结构表征揭示了SSAI中中间螺旋和175环的三种不同构象（向下、向上和超级向上），以及SSRI的一种晶体形式中该区域的完全无序状态，这表明这些变体受损的亲和力与构象灵活性有关。研究了因子Xa中配体结合位点周边的谷氨酸217的影响。将谷氨酸217引入含有SSFI序列的胰蛋白酶变体中，对因子Xa配体（2）和（3）表现出增强的亲和力。这些变体的晶体结构也呈现出向下和超级向上构象，后者在浸泡和结合抑制剂（2）后可转变为向上构象。含谷氨酸217变体亲和力的提高似乎是由于向向上构象的转变。因此，由蛋白质靶点构象变异性引起的亲和力降低可以通过与向上构象的补偿性结合部分或完全抵消。这些研究提供的见解将有助于增进我们对药物设计过程中配体结合的理解。
