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

Authors :
Rayaprolu V
Miettinen HM
Baker W
Young VC
Fisher M
Mueller G
Rankin WO
Kelley JJ
Ratzan W
Leong LM
Davisson JA
Baker BJ
Kohout SC
Source :
BioRxiv : the preprint server for biology [bioRxiv] 2024 Apr 09. Date of Electronic Publication: 2024 Apr 09.
Publication Year :
2024

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 as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, 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 were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine 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 enzymes conformational response to membrane potential transients and influencing the function of the VSD.

Details

Language :
English
Database :
MEDLINE
Journal :
BioRxiv : the preprint server for biology
Accession number :
38234747
Full Text :
https://doi.org/10.1101/2023.12.27.573443