Structural Characterization of the Full Length CrgA Protein Found in Mycobacterium Tuberculosis

Abstract

Mycobacterium Tuberculosis, or Mtb is a pathogen of Tuberculosis. Mtb has a unique stage called latency, when Mtb temporarily shuts down cell division. During this stage, antibiotics are not able to kill the bacteria, so a treatment has to continue for 6 months. Today multidrug resistant Mtb even necessitates a much longer treatment, up to 2 years. The development of new drugs is critically important.CrgA plays a pivotal role in the Mtb cell division apparatus, the divisome. It is known to recruit and interact with other Mtb proteins associated with cell division. Recently, the transmembrane domain structure of CrgA has been determined using solid state NMR spectroscopy. The goal of this work is to characterize a high resolution structure of CrgA and then characterize CrgA protein-protein complexes with a view toward drug development. In addition, the N-terminus and the interhelical loop need to be characterized to identify additional protein binding sites.The full length CrgA is overexpressed in E. coli yielding 40mg per liter of culture. The purified protein is reconstituted in the synthetic lipid bilayers to induce the native structure. To define the secondary structure of the N-terminus and the interhelical loop, two different 2D magic angle spinning solid state NMR experiments are performed with specific amino acid labeling strategies. DARR experiments provide the information about 13C chemical shifts and 13C-13C correlations and both 13C & 15N isotropic chemical shifts are obtained from NCA experiments. Recently, it has been recognized that CrgA functions as a dimer based on bacterial two hybrid assays. From an analysis of the membrane exposed glycine residues, binding interfacial sites for the dimer-dimer interface have been suggested as well as possible sites for interaction for its protein recruitment partners.