Gauging a hydrocarbon ruler by an intrinsic exciton probe
Gauging a hydrocarbon ruler by an intrinsic exciton probe
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DOI：
10.1021/bi602526k
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发表时间：
2007-04-17
期刊：
影响因子：
2.9
通讯作者：
Bishop, Russell E.
中科院分区：
文献类型：
--
作者：
Khan, M. Adil;Neale, Chris;Bishop, Russell E.
膜内酶选择脂质酰基链的结构基础尚不清楚，因为大多数脂质代谢的整体膜酶已被证明难以确定结构；然而，来自革兰氏阴性菌外膜的强大酶现在提供了对潜在机制的初步了解。磷脂：脂质A棕榈酰基转移酶PagP的亚甲基单位分辨率由碳氢标尺决定，碳氢标尺是埋在跨膜β桶结构中的16碳饱和酰基链结合口袋。在烃尺底部用Ala或Met取代Gly88，使酶分别选择15碳或12碳饱和的酰基链，表明烃尺深度决定了酰基链的选择。然而，Gly88Cys PagP的分辨率不会线性降低，因为它同时选择14碳和15碳饱和酰基链。我们发现，埋藏的Tyr26-Trp66苯酚-吲哚相互作用产生的激子被近端Gly88Cys PagP巯基引起的局部结构扰动所熄灭。单个Cys的位点特异性s-甲基化产生了gly88cys -s -甲基PagP，通过特异性地选择13碳饱和酰基链转移到脂质a，重新确定了激子和亚甲基单元的分辨率。与其他Gly88取代不同，Cys巯基从烃基底部退下，局部干扰下的Tyr26和Trp66芳环。由此产生的烃标尺膨胀以激子为代价发生，并在选定的酰基链中容纳一个额外的亚甲基单位。烃类标尺和激子的并置使PagP成为一种分子尺度，用于探索膜内环境中脂质酰基链选择的结构基础。
The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of Gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.