Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase

J Gen Physiol. 2024 Jul 1;156(7):e202313467. doi: 10.1085/jgp.202313467. Epub 2024 May 21.

Abstract

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

MeSH terms

  • Animals
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Mutation
  • Phosphoric Monoester Hydrolases* / chemistry
  • Phosphoric Monoester Hydrolases* / genetics
  • Phosphoric Monoester Hydrolases* / metabolism
  • Phosphorylation
  • Protein Domains
  • Xenopus Proteins / chemistry
  • Xenopus Proteins / metabolism
  • Xenopus Proteins / physiology
  • Xenopus laevis

Substances

  • Phosphoric Monoester Hydrolases
  • voltage-sensor-containing phosphatase, Ciona intestinalis
  • Xenopus Proteins