Interfacial binding of bee venom secreted phospholipase A2 to membranes occurs predominantly by a nonelectrostatic mechanism
Interfacial binding of bee venom secreted phospholipase A2 to membranes occurs predominantly by a nonelectrostatic mechanism
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DOI：
10.1021/bi049390i
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发表时间：
2004-10-26
期刊：
影响因子：
2.9
通讯作者：
Gelb, MH
中科院分区：
文献类型：
--
作者：
Bollinger, JG;Diraviyam, K;Gelb, MH
蜂毒分泌型磷脂酶A（2）（bvPLA（2））含有一个主要由疏水残基和两个与膜紧密接触的碱性残基组成的膜结合表面。先前的研究表明，突变体中这两个碱性残基（K14和R23）以及附近的其他三个碱性残基共同改变为谷氨酸（电荷反转），像野生型酶一样，以高亲和力结合阴离子磷脂囊泡。在本研究中，我们测量了野生型bvPLA（2）、电荷反转突变体（bvPLA（2）-E5）和其中5个碱性残基变为中性谷氨酰胺的突变体（bvPLA（2）-Q5）与含有不同量的阴离子磷脂酰丝氨酸（PS）的磷脂酰胆碱（PC）囊泡的相互作用的平衡常数。值得注意的是，随着PS摩尔百分比的增加，具有阴离子膜结合表面的bvPLA（2）-E5更紧密地结合囊泡。计算研究预测，这是由于当酶结合PC/PS囊泡时，E14（和在一定程度上E23）的pK（a）显著上移，使得膜表面附近的谷氨酸侧链的羧酸盐经历质子化。囊泡结合的实验pH依赖性支持这一预测。bvPLA（2）-E5与PS/PC囊泡的结合比野生型酶弱，这是由于静电蛋白质-囊泡排斥以及伴随酶-囊泡接触的碱性残基和谷氨酸盐的去溶剂化的类似能量学。对bvPLA（2）-Q5的研究表明，总的bvPLA（2）界面结合能中只有一小部分（约10%）是由静电引起的。
The secreted phospholipase A(2) from bee venom (bvPLA(2)) contains a membrane binding surface composed mainly of hydrophobic residues and two basic residues that come in close contact with the membrane. Previous Studies have shown that the mutant in which these two basic residues (K14 and R23) as well as three other nearby basic residues were collectively changed to glutamate (charge reversal), like wild-type enzyme, binds with high affinity to anionic phospholipid vesicles. In the present study, we have measured the equilibrium constants for the interaction of wild-type bvPLA(2), the charge-reversal mutant (bvPLA(2)-E5), and the mutant in which the five basic residues were changed to neutral glutamine (bvPLA(2)-Q5) with phosphatidylcholine (PC) vesicles containing various amounts of the anionic phosphatidylserine (PS). Remarkably, bvPLA(2)-E5 with an anionic membrane binding surface binds more tightly to vesicles as the mole percent of PS is increased. Computational studies predict that this is due to a significant upward shift in the pK(a) of E14 (and to some extent E23) when the enzyme binds to PC/PS vesicles such that the carboxylate of the glutamate side chain near the membrane surface undergoes protonation. The experimental pH dependence of vesicle binding supports this prediction. bvPLA(2)-E5 binds more weakly to PS/PC vesicles than does wild-type enzyme due to electrostatic protein-vesicle repulsion coupled with the similar energetics of desolvation of basic residues and glutamates that accompanies enzyme-vesicle contact. Studies with bvPLA(2)-Q5 show that only a small fraction of the total bvPLA(2) interfacial binding energy(similar to10%) is due to electrostatics.