Solution Structure of the Conserved Hypothetical Protein Rv2302 from Mycobacterium tuberculosis

The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel -sheet core with one C-terminal -helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the -helix and -strands 3 and 4 hold the -helix near the -sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state { 1 H}- 15 N heteronuclear nuclear Overhauser effects indicate that the protein’s core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the 1 H- 15 N heteronuclear single quantum correlation spectrum suggests that this region, a loop between -strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold. Mycobacterium tuberculosis is the etiological agent responsible for the chronic infectious disease tuberculosis. Despite the availability of effective short-term chemotherapy and extensive vaccination programs, this gram-positive tubercle bacillus still infects approximately one-third of mankind, and in 2003 it is estimated that it claimed the lives of approximately 1.7 million people (44). Indeed, there has been a recent increase in the incidence of tuberculosis in both developing and industrialized nations as new drug-resistant strains have emerged, and a deadly synergy with human immunodeficiency virus has evolved (17). The complete sequenced genome of M. tuberculosis contains approximately 4,000 genes (6). In 2000 a TB Structural Genomics Consortium was established with the goal of using the genomic information to discover and analyze the structures of the M. tuberculosis gene products (38). In addition to providing a foundation for a fundamental understanding of biology, the information and knowledge obtained from determining the structures of the proteins in the M. tuberculosis genome will enable the conception and development of new therapies and strategies to treat and control this deadly disease. In pursuit of these goals, we have used nuclear magnetic resonance (NMR)-based methods to determine the solution structure for a protein, Rv2302, that is highly conserved in the bacterium M. tuberculosis.