Novakova Zora, Cerny Jiri, Choy Cindy J, Nedrow Jessie R, Choi Joeseph K, Lubkowski Jacek, Berkman Clifford E, Barinka Cyril
Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Department of Chemistry, Washington State University, Pullman, WA, USA.
FEBS J. 2016 Jan;283(1):130-43. doi: 10.1111/febs.13557. Epub 2015 Nov 5.
Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds.
Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).
靶向人谷氨酸羧肽酶II（GCPII）的抑制剂通常由一个源自P1'谷氨酸的结合模块组成，该模块保证了高亲和力和特异性，并与位于酶入口通道内的效应功能相连。在此，我们进行了一项全面的结构和计算研究，旨在剖析效应功能对GCPII结合和亲和力的重要性。为此，我们确定了人GCPII与一系列具有不同物理化学特性效应功能的氨基磷酸酯类抑制剂复合物的晶体结构。我们的数据表明，与匹配的膦酸酯相比，氨基磷酸酯的更高结合亲和力与酶的Glu424和Gly518与氨基磷酸酯的酰胺基团之间存在额外的氢键有关。虽然源自P1'谷氨酸的模块在GCPII的S1'口袋中的定位是不变的，但效应功能与入口通道内衬残基之间的相互作用界面高度不同，由Arg463、Arg534和Arg536定义的带正电荷的精氨酸区域是几个研究复合物共有的唯一“热点”。这种变异性部分源于这样一个事实，即效应器/GCPII界面通常包含入口通道内非极性残基的孤立区域，并且由此产生的范德华接触缺乏氢键相互作用典型的方向性。所呈现的数据揭示了GCPII非prime位点内抑制剂结合模式的复杂性，可用于设计新型GCPII特异性化合物。
PDB ID代码：本结构的原子坐标以及实验结构因子振幅已存入RCSB蛋白质数据库，登录代码为4P44（与JRB - 4 - 81的复合物）、4P45（与JRB - 4 - 73的复合物）、4P4B（与CTT54的复合物）、4P4D（与MP1C的复合物）、4P4E（与MP1D的复合物）、4P4F（与NC - 2 - 40的复合物）、4P4I（与T33的复合物）和4P4J（与T33D的复合物）。
