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...

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Bibliographic Details
Published in:Protein science 2024-06, Vol.33 (6), p.e5009-n/a
Main Authors: Zavala, Erik, Dansereau, Stephen, Burke, Michael J., Lipchock, James M., Maschietto, Federica, Batista, Victor, Loria, J. Patrick
Format: Article
Language:English
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
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Summary: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.
ISSN:0961-8368
1469-896X
1469-896X
DOI:10.1002/pro.5009