Structure-Function Studies of Mtb Membrane Protein CrgA in Lipid Bilayer

Abstract

Tuberculosis is a deadly disease with a death toll of 1.5 million people every year and very recently an outbreak in Jacksonville, FL was devastating. All frontline antibiotics are failed for the multidrug resistant (MDR) bacilli and the key to disease control is to inhibit bacterial cell division and stabilize the bacilli in its dormant stage. Here we present the structure function studies of a novel drug target membrane protein CrgA in M. tuberculosis peptidoglycan polymerization complex. We show that CrgA interacts strongly with FtsZ, FtsQ, CwsA and the class B penicillin binding proteins such as FtsI and PBPA in M. smegmatis. CwsA a binding partner of CrgA binds to Wag31 another key protein in peptidoglycan synthesis. Co-localization of CrgA with FtsZ in the mid cell region facilitates the Z-ring formation during the cell division process. Thus structural characterization of CrgA in a lipid bilayer is essential. Full length CrgA has 2 transmembrane (TM) helices, a 32 residue N terminal domain, a very short C terminal domain, and a 10 residue inter-helical loop for a total of 93 residues and an 11.4 kDa molecular weight. Full length 15N, 13C uniform and amino acid specific labeled CrgA was over-expressed and purified from E. coli with a yield of 35 mg/L. To mimic the membrane environment, isotope labeled CrgA was reconstituted in POPC:POPG liposomes. Orientation dependent 2D Polarization Inversion Spin Exchange at Magic Angle (PISEMA) and 3D Magic Angle Spinning (MAS) solid state NMR experiments were performed to obtain structural restraints. For all ssNMR experiments Low E probes in 600 and 900 MHz magnets were used at the NHMFL. CrgA TMD structure was calculated by CS-Rosetta and XPLOR-NIH. Structure refinement was enhanced by restrained molecular dynamics in a lipid bilayer environment.