Structural and Functional Characterization of the Mycobacterium Tuberculosis Virulence Factor Protein Tyrosine Phosphatase B (Ptpb)

The Mycobacterium tuberculosis genome encodes two protein tyrosine phosphatase (PTP) enzymes, PtpA and PtpB, which are secreted virulence factors that dephosphorylate host proteins in order help the bacterium achieve immune escape. Despite low sequence identity (20% from bacteria to humans) amongst the PTP superfamily, these proteins have a conserved structural fold. Structural characterizations of Mtb PtpB enzyme reveal the same core structural fold, but two unique structural insertions corresponding to a two-helix "lid" that blocks the active site and additional helix (helix alpha3a) that undergoes an order-disorder transition. The protein is clearly active in solution, but inactive in crystals leading the hypothesis that the lid must open and close for catalysis to occur. Additionally, biochemical oxidative inactivation studies of the catalytic cysteine residue demonstrated that the PtpB protein is more resistant to oxidative inactivation than other PTPs lacking a lid. Using these observations and the static crystal structures as a molecular guide, I sought to characterize these unique features' dynamics and how they contribute to the function of the PtpB enzyme. Single Molecule FRET (SM FRET) was used as a spectroscopic ruler to provide first direct evidence of lid opening and closing on the biologically relevant ms time scale in both helices of the PtpB lid (helix alpha7, alpha8). The rates and populations of open and closed conformers were obtained from the experimental analysis, which showed that the both helices of the lid are closed approximately 70% of the time. Assuming that lid closure excludes oxidant, the rates of opening and closing quantitatively accounted for the rate of oxidative inactivation. Increasing solvent viscosity by the addition of glycerol but not PEG8000, resulted in higher rates of oxidative inactivation, and SM FRET analysis demonstrated that this was due to an increase in the population of open conformers. We measured different rates of lid motions for the two helices of the lid, supporting the idea that these helices can move asynchronously. These differences can be explained by an interference of the disorder-order transition of helix alpha3a, which folds upon the protein and displaces helix alpha8 in its closed conformation. We dissected the PtpB structure with site directed mutagenesis, which resulted in classification of the specific interactions in the lid, helix alpha3a, and protein core that are important for substrate binding, catalysis, lid dynamics, and helix alpha3a dynamics. More specifically, we found that perturbation of the electrostatic environment of the active site cavity reduces the kinetic parameters of the PtpB enzyme. We observed a connection between lid dynamics and helix alpha3a dynamics, but we found that the dynamics that occur during catalysis are not necessarily the same as those that occur in the absence of substrates. Additionally, we found two specific residues in the helix 3a that contribute to helix stability and the disorder-order transition. Finally, we determined the location of the protein hinge that controls lid movements and that packing of this hinge region controls the rates of lid dynamics.