Characterization of an Intrinsic Disorder Domain and Functional Activity of ChiZ Membrane Protein from Mycobacterium tuberculosis

Abstract

ChiZ is a membrane protein from Mycobacterium tuberculosis (MTB) that is involved in cell division. This protein has an intracellular domain that is known to be intrinsically disordered. Interestingly, this cytoplasmic domain is able to hydrolyze peptidoglican (PG) in in vitro assays. ChiZ also contains a C-terminal LysM domain for PG binding in the periplasm. ChiZ is an interesting target for structural characterization and drug development because it is involved in cell division.This work mainly focuses on the nascent structural characterization of the intrinsically disorder region of ChiZ (ChiZ1-64) using solution NMR in addition to biochemical assays for understanding its function. Biochemical assays indicate that ChiZ1-64 is able to hydrolyze PG at pH 4.0 and 7.0. As expected, solution NMR experiments of ChiZ1-64 show a typical spectrum of an unfolded protein. However, HSQC spectra at these two pHs indicate the presence of two distinct regions with different backbone dynamics and signal intensity. Sequence alignment of ChiZ homologs found in other species of Mycobacterium also shows two different regions, one of variable length (residues 1 to 37 in MTB), and another conserved region (residues 38 to 64 in MTB). Both conserved and variable regions match to the regions identified with solution NMR experiments. Removal of the variable region in ChiZ produces an active protein (ChiZ38-165) at low pH. This suggests that the conserved region is enough to hydrolyze PG. However, the variable region appears to be important for full activity at neutral pH. Additional binding assays of ChiZ1-64 show that this region is able to bind PG. This is demonstrated by the depletion of the protein from solution and the detection of protein using solid state NMR of PG and ChiZ1-64 in complex.