title: Glutamate 85 and glutamate 228 contribute to the pH-response of the soluble form of chloride intracellular channel 1.
abstract: The chloride intracellular channel protein, CLIC1, is synthesised as a soluble monomer that can reversibly bind membranes. Soluble CLIC1 is proposed to respond to the low pH found at a membrane surface by partially unfolding and restructuring into a membrane-competent conformation. This transition is proposed to be controlled by strategically located "pH-sensor" residues that become protonated at acidic pH. In this study, we investigate the role of two conserved glutamate residues, Glu85 in the N-domain and Glu228 in the C-domain, as pH-sensors. E85L and E228L CLIC1 variants were created to reduce pH sensitivity by permanently breaking the bonds these residues form. The structure and stability of each variant was compared to the wild type at both pH 7.0 and pH 5.5. Neither substitution significantly altered the structure but both decreased the conformational stability. Furthermore, E85L CLIC1 formed a urea-induced unfolding intermediate state at both pH 7 and pH 5.5 compared to wild-type and E228L CLIC1 which only formed the intermediate at pH 5.5. We conclude that Glu85 and Glu228 are two of the five pH-sensor residues of CLIC1 and contribute to the pH-response in different ways. Glu228 lowers the stability of the native state at pH 5.5, while Glu85 contributes both to the stability of the native state and to the formation of the intermediate state. By putting these interactions into the context of the three previously described CLIC1 pH-sensor residues, we propose a mechanism for the conversion of CLIC1 from the soluble state to the pre-membrane form.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, 1 Jan Smuts Ave, Johannesburg, 2050, South Africa.
authors.forename: Megan
authors.initials: M
authors.lastname: Cross
authors.forename: Manuel
authors.initials: M
authors.lastname: Fernandes
authors.forename: Heinrich
authors.initials: H
authors.lastname: Dirr
authors.forename: Sylvia
authors.initials: S
authors.lastname: Fanucchi
mesh_terms: D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000494:pharmacology
chemical_list: C501966:CLIC1 protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D018698:Glutamic Acid; D014508:Urea