Pan Ying H, Yu Bao-Zhu, Berg Otto G, Jain Mahendra K, Bahnson Brian J
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, and Department of Molecular Evolution, Uppsala University Evolutionary Biology Center, Uppsala, Sweden.
Biochemistry. 2002 Dec 17;41(50):14790-800. doi: 10.1021/bi026922r.
We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.
我们解析了猪胰脏I B族磷脂酶A2（PLA2）的阴离子辅助二聚体的晶体结构，分辨率为1.55 Å，该二聚体与底物血小板活化因子的水解产物复合。二聚体在两个PLA2亚基之间的接触面含有五个共面的磷酸根阴离子。此结构与先前报道的模拟PLA2与底物界面结合的四面体中间体的阴离子辅助二聚体相似[Pan, Y. H., 等人（2001年）《生物化学》40, 609 - 617]。二聚体结构中，产物乙酸分子结合在亚基A中，另一个产物1 - 十八烷基 - sn - 甘油 - 3 - 磷酸胆碱（LPC - 醚）结合在亚基B中。因此，此结构是两个单独的产物二元复合物，而非两个产物都在PLA2一个活性位点的三元复合物。仅通过从初始底物开始共结晶才能获得结合有产物的蛋白质晶体。相比之下，当PLA2与LPC - 醚和琥珀酸盐共结晶时，得到了另一种晶体形式，且这种晶体形式不包含结合的产物。产物结合结构中，乙酸位于亚基A的催化位点，其一个氧原子距离催化钙3.5 Å。同样，钙与甘氨酸（32）羰基的距离为3.4 Å，长于典型距离，导致最终钙的配位为四配位且几何形状扭曲。乙酸的另一个氧原子与N(δ)(1) - 组氨酸（48）、O(δ)(1) - 天冬氨酸（49）以及催化辅助水（w7）形成氢键。相比之下，亚基B中LPC - 醚的甘油磷酸胆碱头部基团与钙或催化残基组氨酸（48）或天冬氨酸（49）没有接触。LPC - 醚的尾部位于活性位点口袋附近，sn - 1 - 酰基链的最后九个碳原子以两种交替构象精修。LPC - 醚产物的其余原子已被构建到溶剂通道中，但由于无序，在精修模型中其占有率设为零。总之，两种产物的晶体学和平衡结合结果表明，两种产物在单个活性位点同时结合是不利的。
