title: Coupling of Ci-VSP modules requires a combination of structure and electrostatics within the linker.
abstract: The voltage-sensitive phosphatase Ci-VSP consists of an intracellular phosphatase domain (PD) coupled to a transmembrane voltage-sensor domain (VSD). Depolarization triggers the selective dephosphorylation of phosphoinositides. However, the molecular mechanisms of coupling are still elusive. To clarify the role of the VSD-PD linker as a putative partner for electrostatic interactions with the membrane, we carried out a cysteine-scanning mutagenesis of the whole motif M240-K257. Upon coexpression with PI(4,5)P(2)-sensitive KCNQ2/KCNQ3 channels in Xenopus oocytes, we identified four positions (A242C, R245C, K252C, and Y255C) with a completely abrogated PD activity. Because the mutation effect occurred periodically, we hypothesize that α-helical elements exist within the linker, with a gap near position S249. The combination of these results with the analysis of transient sensing currents of the VSD revealed distinct roles for the N-terminal (M240-S249) and C-terminal (Q250-K257) linker motifs in the VSD-PD coupling. According to our functional results, the computational structure prediction of the Q239-D258 fragment confirmed α-helical structures within the linker, with a short β-turn around S249 in the activated conformation. Remarkably, the position K252 may be a candidate for interacting with the PD rather than for binding to the membrane. This provides the first insight (to our knowledge) into the direct intervention of the linker in the VSD-PD coupling process.
authors.affiliation: Berlin Institute of Technology, Institute of Chemistry, Max-Volmer-Laboratory of Biophysical Chemistry, Berlin, Germany. kirstin.hobiger@tu-berlin.de
authors.forename: Kirstin
authors.initials: K
authors.lastname: Hobiger
authors.forename: Tillmann
authors.initials: T
authors.lastname: Utesch
authors.forename: Maria Andrea
authors.initials: MA
authors.lastname: Mroginski
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D015640:Ion Channel Gating; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: D050505:Mutant Proteins; C509671:voltage-sensor-containing phosphatase, Ciona intestinalis; D010744:Phosphoric Monoester Hydrolases; D003545:Cysteine