期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：2
页码：E119-E126
DOI：10.1073/pnas.1415908112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceUnderstanding the structure and function of the cell division apparatus of Mycobacterium tuberculosis is crucial for advancing drug development against tuberculosis. Here, we report the solid-state NMR structure of a transmembrane protein, CrgA, that is a central component of the M. tuberculosis divisome. Small helical membrane protein structures are particularly sensitive to their environment, and consequently, we characterized CrgA in an environment that models well the biophysical properties of the native membrane. To determine the structure, both oriented sample and magic-angle spinning NMR data from liquid-crystalline lipid bilayer preparations were used along with refinement by restrained molecular dynamics simulations in the same lipid environment. The structure suggests how CrgA serves as a platform for binding and recruiting other proteins of the divisome. The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid-crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24{degrees} at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA's partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines.
