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

• Published in: Protein science Vol. 33; no. 6; pp. e5009 - n/a
• Main Authors: Zavala, Erik, Dansereau, Stephen, Burke, Michael J., Lipchock, James M., Maschietto, Federica, Batista, Victor, Loria, J. Patrick
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.06.2024; Wiley Subscription Services, Inc
• Subjects: Affinity; Binding; Carboxyl group; catalysis; Catalytic activity; Catalytic Domain; Chemical equilibrium; Dynamic structural analysis; Electrostatic properties; electrostatics; Eukaryotes; Glycine; Histidine; Humans; Molecular dynamics; Molecular Dynamics Simulation; molecular dynamics simulations; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; phosphatases; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - metabolism; Salts; structural dynamics; Substrate preferences; Substrate Specificity; Substrates; molecular dynamics simulations; catalysis; NMR; electrostatics; structural dynamics; phosphatases
• ISSN: 0961-8368; 1469-896X; 1469-896X
• DOI: 10.1002/pro.5009

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.