Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.
The Mycobacterium tuberculosis protein tyrosine phosphatase PtpB shows resistance to the oxidative conditions that prevail within an infected host macrophage, but the mechanism of this molecular adaptation is unknown. Crystal structures of PtpB revealed previously that a closed, two-helix lid covers the active site. By measuring single-molecule Forster-type resonance energy transfer to probe the dynamics of two helices that constitute the lid, we obtained direct evidence for large, spontaneous opening transitions of PtpB with the closed form of both helices favored approximately 3:1. Despite similar populations of conformers, the two helices move asynchronously as demonstrated by different opening and closing rates under our experimental conditions. Assuming that lid closure excludes oxidant, the rates of opening and closing quantitatively accounted for the slow observed rate of oxidative inactivation. Increasing solvent viscosity using glycerol but not PEG8000 resulted in higher rates of oxidative inactivation due to an increase in the population of open conformers. These results establish that the rapid conformational gating of the PtpB lid constitutes a reversible physical blockade that transiently masks the active site and retards oxidative inactivation.
Department of Molecular and Biology and QB3 Institute, University of California, Berkeley, California 94720, USA.
E Megan
EM
Flynn
Jeffrey A
JA
Hanson
Tom
T
Alber
Haw
H
Yang
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
D006861:Hydrogen Peroxide; D017027:Protein Tyrosine Phosphatases