S1 hydrophobic residues modulate voltage sensing phosphatase enzymatic function and voltage sensing

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Voltage sensing domains (VSDs) convert electrical signals to conformational changes.
• The S1-S3 helices typically act as scaffolding, except in voltage sensing phosphatases (VSPs) and proton channels (Hv).
• VSPs are voltage-regulated enzymes, while Hvs are channels composed solely of VSDs.

Approach:

• Investigated the S1 helix contribution to VSP function by creating single and quadruple alanine mutations.
• Assessed the impact of mutations on voltage dependence, enzymatic kinetics, and VSD motions.
• Utilized co-immunoprecipitation to examine VSP dimer integrity and introduced mutations into a voltage indicator to study optical readings.

Key Points:

• Mutations in the S1 helix shifted the voltage dependence of VSP activity to higher voltages, altering substrate reaction kinetics and slowing dephosphorylation.
• VSD motions exhibited voltage dependence shifted to lower voltages, suggesting a second voltage-dependent motion.
• Mutations did not disrupt VSP dimer formation, indicating intrasubunit and/or intersubunit interactions mediate the S1 helix's impact.
• Introducing S1 mutations into a voltage indicator significantly altered optical readings, affecting kinetics and voltage dependence.

Conclusions:

• The S1 helix in VSPs is critical for tuning the enzyme's conformational response to membrane potential.
• S1 helix modifications influence VSD function and the overall activity of voltage sensing proteins.
• This study highlights the S1 helix as a key regulatory element in VSP function and VSD dynamics.