Chandra V, Kaur P, Srinivasan A, Singh T P
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, 110 029, India.
J Mol Biol. 2000 Mar 3;296(4):1117-26. doi: 10.1006/jmbi.2000.3537.
The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.
圆斑蝰蛇磷脂酶A₂（DPLA₂）是已知的IIA组II亚类的唯一成员。已通过分子置换法，以2.4埃的分辨率确定了这种突触前神经毒性DPLA₂酶的三维结构。使用矛头蝮模型通过分子置换法确定结构，并使用X-PLOR进行精修，对于分辨率范围在20.0埃至2.4埃的所有数据，最终R因子为18.8%。最终精修模型包含来自两个晶体学独立蛋白质分子的1888个原子和160个水氧原子。DPLA₂的整体折叠结构有三个长螺旋和两条短的反平行β链，与其他磷脂酶A₂的结构大体相似。在当前结构中，钙结合位点为空，但钙结合环的构象与在钙结合状态下观察到的相似。55 - 61位残基（典型的β-转角I）和83 - 94位残基（明确的环）的两个空间相邻区域，在构象、静电特性和片段间相互作用方面，与非神经毒性磷脂酶A₂中的情况显著不同。DPLA₂的另一个显著结构特征与53 - 77位残基序列有关，该序列有一系列带正电荷的残基向外突出。上述片段被认为与抗凝活性有关。在表面发现了一个独特的疏水斑块，包括Leu17、Ala18、Ile19、Pro20、Phe106和Leu110等残基，以及一个同样明显的含-OH基团的区域，包含Ser21、Tyr22、Ser23、Ser24、Tyr25和Tyr28等残基。不对称单元中两个DPLA₂分子之间的相互作用与磷脂酶A₂的标准二聚体和三聚体中观察到的显著不同，使酶的活性位点完全暴露于酶-底物反应，这使得该结构成为通过浸泡实验进行基于结构的药物设计的最有利实例之一。
