Hubbard S J, Eisenmenger F, Thornton J M
Department of Biochemistry and Molecular Biology, University College, London, United Kingdom.
Protein Sci. 1994 May;3(5):757-68. doi: 10.1002/pro.5560030505.
Previous analyses of limited proteolytic sites within native, folded protein structures have shown that a significant conformational change is required in order to facilitate binding into the active site of the attacking proteinase. For the serine proteinases, the optimum conformation to match the proteinase binding-site geometry has been well characterized crystallographically by the conserved main-chain geometry of the reactive site loops of their protein inhibitors. A good substrate must adopt a conformation very similar to this "target" main-chain conformation prior to cleavage. Using a "loop-closure" modeling approach, we have tested the ability of a set of tryptic-limited proteolytic sites to achieve this target conformation and further tested their suitability for cleavage. The results show that in most cases, significant changes in the conformation of at least 12 residues are required. All the putative tryptic cleavage sites in 1 protein, elastase, were also modeled and tested to compare the results to the actual nicksite in that protein. These results strongly suggest that large local motions proximate to the scissile bond are required for proteolysis, and it is this ability to unfold locally without perturbing the overall protein conformation that is the prime determinant for limited proteolysis.
先前对天然折叠蛋白质结构内有限蛋白水解位点的分析表明，为了便于结合到攻击蛋白酶的活性位点，需要显著的构象变化。对于丝氨酸蛋白酶，通过其蛋白质抑制剂反应位点环的保守主链几何结构，在晶体学上已经很好地表征了与蛋白酶结合位点几何结构相匹配的最佳构象。一个好的底物在切割之前必须采用与这种“靶标”主链构象非常相似的构象。使用“环闭合”建模方法，我们测试了一组胰蛋白酶有限蛋白水解位点实现这种靶标构象的能力，并进一步测试了它们的切割适用性。结果表明，在大多数情况下，至少需要12个残基的构象发生显著变化。还对一种蛋白质弹性蛋白酶中的所有假定胰蛋白酶切割位点进行了建模和测试，以将结果与该蛋白质中的实际切口位点进行比较。这些结果强烈表明，蛋白水解需要靠近可裂解键的大量局部运动，而正是这种在不干扰整体蛋白质构象的情况下局部展开的能力是有限蛋白水解的主要决定因素。
