Dynamic Active-Site Protection by the M. tuberculosis Protein Tyrosine Phosphatase PtpB Lid Domain

摘要

The Mycobacterium tuberculosis protein tyrosine phosphatase PtpB shows resistance to thenoxidative conditions that prevail within an infected host macrophage, but the mechanism of this molecularnadaptation is unknown. Crystal structures of PtpB revealed previously that a closed, two-helix lid coversnthe active site. By measuring single-molecule Fo¨ rster-type resonance energy transfer to probe the dynamicsnof two helices that constitute the lid, we obtained direct evidence for large, spontaneous opening transitionsnof PtpB with the closed form of both helices favored ∼3:1. Despite similar populations of conformers, thentwo helices move asynchronously as demonstrated by different opening and closing rates under ournexperimental conditions. Assuming that lid closure excludes oxidant, the rates of opening and closingnquantitatively accounted for the slow observed rate of oxidative inactivation. Increasing solvent viscositynusing glycerol but not PEG8000 resulted in higher rates of oxidative inactivation due to an increase in thenpopulation of open conformers. These results establish that the rapid conformational gating of the PtpB lidnconstitutes a reversible physical blockade that transiently masks the active site and retards oxidativeninactivation.