A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

MptpA Kinetics Enhanced by Allosteric Control of an Active Conformation.

Understanding allostery in the Mycobacterium tuberculosis low molecular weight protein tyrosine phosphatase (MptpA) is a subject of great interest since MptpA is one of two protein tyrosine phosphatases (PTPs) from the pathogenic organism Mycobacterium tuberculosis expressed during host cell infection. Here, we combine computational modeling with solution NMR spectroscopy and we find that Q75 is an allosteric site. Removal of the polar side chain of Q75 by mutation to leucine results in a cascade of events that reposition the acid loop over the active site and relocates the catalytic aspartic acid (D126) at an optimal position for proton donation to the leaving aryl group of the substrate and for subsequent hydrolysis of the thiophosphoryl intermediate. The computational analysis is consistent with kinetic data, and NMR spectroscopy, showing that the Q75L mutant exhibits enhanced reaction kinetics with similar substrate binding affinity. We anticipate that our findings will motivate further studies on the possibility that MptpA remains passivated during the chronic state of infection and increases its activity as part of the pathogenic life cycle of M. tuberculosis possibly via allosteric means.