Fang Bin, Fu Guoxing, Agniswamy Johnson, Harrison Robert W, Weber Irene T
Department of Biology, Molecular Basis of Disease Program, Georgia State University, Atlanta, GA 30303, USA.
Apoptosis. 2009 May;14(5):741-52. doi: 10.1007/s10495-009-0333-y.
Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9-2.6 A. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.
通过晶体学、动力学以及对模型复合物的计算，研究了半胱天冬酶 -3对其众多蛋白质底物中各种P4残基的识别。天冬氨酸是肽底物中最常见的P4残基，尽管在被半胱天冬酶 -3切割的细胞蛋白质中发现了各种各样的P4残基。具有疏水性P4残基或无P4残基的肽类抑制剂的结合情况，通过半胱天冬酶 -3与Ac-IEPD-Cho、Ac-WEHD-Cho、Ac-YVAD-Cho和Boc-D(OMe)-Fmk复合物的晶体结构得以阐明，分辨率为1.9 - 2.6埃。P4残基在结合位点的两个独立疏水区域形成了有利的疏水相互作用。P4异亮氨酸和酪氨酸的侧链与S4亚位点非极性口袋内的半胱天冬酶 -3残基色氨酸206和色氨酸214形成疏水相互作用，而P4色氨酸与也可形成S5亚位点的苯丙氨酸250和苯丙氨酸252相互作用。这些疏水性P4残基的相互作用与极性P4天冬氨酸的相互作用不同，这表明半胱天冬酶 -3对结合不同P4残基具有适应性。分子模型预测的肽结合趋势与肽类抑制剂的实验值具有高度相关性。对醛肽Ac-XEVD-Cho中P4位置20种不同氨基酸结合的结构模型分析表明，大多数亲水性P4残基与苯丙氨酸250相互作用，而疏水性残基与色氨酸206、苯丙氨酸250和色氨酸214相互作用。总体而言，半胱天冬酶 -3的S4口袋对不同残基表现出灵活的适应性，新的结构和模型，特别是针对疏水性P4残基的结构和模型，将有助于基于半胱天冬酶 -3的药物设计。
