Miller Aaron S, Falke Joseph J
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Biochemistry. 2004 Feb 24;43(7):1763-70. doi: 10.1021/bi0360206.
Previous model studies of peptides and proteins have shown that protein-lipid interactions, primarily involving amino acid side chains near the membrane-water interface, modulate the position of transmembrane helices in bilayers. The present study examines whether such interfacial side chains stabilize the signaling states of a transmembrane signaling helix in a representative receptor, the aspartate receptor of bacterial chemotaxis. To examine the functional roles of signaling helix side chains at the periplasmic and cytoplasmic membrane-water interfaces, arginine and cysteine substitutions were scanned through these two interfacial regions. The chemical reactivities of the cysteine residues were first measured to determine the positions at which the helix crosses the membrane-water interface in both the periplasmic and cytoplasmic compartments. Subsequently, two antisymmetric in vitro activity measurements were carried out to determine the effect of each interfacial arginine or cysteine substitution on receptor signaling. Substitutions that stabilize the receptor on-state cause upregulation of receptor-coupled kinase activity and inhibition of methylation at receptor adaptation sites, while substitutions that stabilize the off-state have the opposite effects on these two activities. Notably, four substitutions at aromatic tryptophan and phenylalanine positions buried in the membrane near the membrane-water interface were found to stabilize the native on- or off-signaling state. The striking ability of these substitutions to drive the receptor toward a specific signaling state indicates that interfacial side chains are highly optimized to correctly position the native signaling helix in the membrane and to allow normal switching between the on- and off-signaling states. The analogous substitutions in model transmembrane helices are known to drive small piston-type displacements of the helix normal to the membrane. Thus, the simplest molecular interpretation of the present findings is that the signal-stabilizing substitutions drive piston displacements of the signaling helix, providing further support for the piston model for transmembrane signaling in bacterial chemoreceptors. More generally, the findings indicate that the interfacial phenylalanine, tryptophan, and arginine side chains widespread in the transmembrane alpha-helices of receptors, channels, and transporters can play important roles in modulating transitions between signaling and conformational states.
先前对肽和蛋白质的模型研究表明，蛋白质-脂质相互作用主要涉及膜-水界面附近的氨基酸侧链，可调节双层膜中跨膜螺旋的位置。本研究探讨了这些界面侧链是否能稳定代表性受体（细菌趋化性的天冬氨酸受体）中跨膜信号螺旋的信号状态。为了研究信号螺旋侧链在周质和细胞质膜-水界面的功能作用，在这两个界面区域扫描了精氨酸和半胱氨酸取代。首先测量半胱氨酸残基的化学反应性，以确定螺旋在周质和细胞质区室中穿过膜-水界面的位置。随后，进行了两项不对称的体外活性测量，以确定每个界面精氨酸或半胱氨酸取代对受体信号传导的影响。稳定受体开启状态的取代会导致受体偶联激酶活性上调，并抑制受体适应位点的甲基化，而稳定关闭状态的取代对这两种活性有相反的影响。值得注意的是，发现在膜-水界面附近埋于膜中的芳香族色氨酸和苯丙氨酸位置的四个取代稳定了天然的开启或关闭信号状态。这些取代将受体驱动至特定信号状态的显著能力表明，界面侧链经过高度优化，能在膜中正确定位天然信号螺旋，并允许开启和关闭信号状态之间的正常转换。已知模型跨膜螺旋中的类似取代会驱动螺旋垂直于膜的小活塞式位移。因此，对本研究结果最简单的分子解释是，信号稳定取代驱动信号螺旋的活塞位移，为细菌化学感受器中跨膜信号传导的活塞模型提供了进一步支持。更普遍地说，这些发现表明，广泛存在于受体、通道和转运体跨膜α螺旋中的界面苯丙氨酸、色氨酸和精氨酸侧链在调节信号传导和构象状态之间的转换中可发挥重要作用。
