Szurmant Hendrik, Bunn Michael W, Cho Stephen H, Ordal George W
Department of Biochemistry, Colleges of Medicine and Liberal Arts and Sciences, University of Illinois, Urbana, IL 61801, USA.
J Mol Biol. 2004 Dec 3;344(4):919-28. doi: 10.1016/j.jmb.2004.09.093.
Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.
此前，我们利用二硫键交联对枯草芽孢杆菌化学感受器McpB的跨膜（TM）结构域的组织进行了表征。沿着穿过膜的两条螺旋（TM1和TM2），将半胱氨酸残基工程化到连续位置，以及TM1和TM2中的双突变体，并确定交联程度以表征TM结构域的组织。在本研究中，在有配体和无配体的情况下研究了TM结构域的组织，以探讨发生了哪些配体诱导的结构变化。我们发现，天冬酰胺导致沿着TM1-TM1'螺旋界面的所有残基的交联速率发生变化，而沿着TM2-TM2'界面的几乎所有残基的交联速率没有变化。这些结果表明，在结合天冬酰胺时，螺旋TM1逆时针旋转，而TM2相对于二聚体中的TM2'没有移动。有趣的是，34/280和38/273中配对取代的分子内交联不受天冬酰胺的影响，这表明吸引剂与McpB的结合不会像在大肠杆菌的Trg和Tar中那样诱导TM2的“活塞样”垂直位移。然而，这些配对取代会响应配体产生受体的寡聚形式。这一定是由于在先前提出的二聚体三聚体甚至更高阶复合物中不同受体二聚体之间的界面发生了移动。此外，在天冬酰胺存在下二硫键形成的程度不受甲基修饰酶CheB和CheR或偶联蛋白CheW和CheV的存在的影响，这表明这些蛋白一定对细胞质结构域有局部结构影响，而不会传递到整个受体。最后，二硫键的形成也不受脯氨酸与McpC结合的影响。我们得出结论，配体结合在McpB二聚体的TM结构域中诱导了构象变化，作为一种可能在受体细胞质区域内传播的激发信号，并且随后的适应事件不会影响这种新的TM结构域构象。
