Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli
Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli
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DOI：
10.1021/bi0502801
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发表时间：
2005-05-31
期刊：
影响因子：
2.9
通讯作者：
Kaback, HR
中科院分区：
文献类型：
--
作者：
Ermolova, NV;Smirnova, IN;Kaback, HR
获得大肠杆菌乳糖渗透酶（LacY） x射线结构的关键（Abramson， J., Smirnova， I., Kasho， V., Verner， G., Kaback， H. R.和Iwata， S. (2003) Science 301, 549-716）是使用一种突变体，其中Cys154（螺旋V）被Gly取代。含有这种突变的LacY强烈倾向于一种面向内的构象，这种构象与配体具有高亲和力，但催化很少的运输，并且很少表现出与野生型LacY观察到的依赖于配体的构象变化。x射线结构表明，螺旋V与螺旋I在膜的大约中间交叉，使得Cys154靠近Gly24（螺旋1）。因此，用Gly替换Cys154可能会导致螺旋I和V之间更紧密的包装，从而导致观察到的表型。在C154G突变体中，用Cys替换Gly24可以显著恢复运输活性，并且该突变体在底物结合和热稳定性方面表现出与野生型LacY相似的特性。然而，在某种程度上拯救运输的其他替代品是Val和Asp，它们都比Cys效率低得多。结果表明，尽管螺旋填充可能对C154G突变体的特性起着重要作用，但24位Cys挽救C154G转运活性的能力比24和154位之间简单的散装替换要复杂得多。相反，活性依赖于螺旋之间更微妙的相互作用，而破坏螺旋IV和环6-7之间或螺旋II和IV之间相互作用的突变也可以挽救C154G突变体的运输。
A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix 1). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.