title: Effect of SLC26 anion transporter disease-causing mutations on the stability of the homologous STAS domain of E. coli DauA (YchM).
abstract: The human solute carrier 26 (SLC26) family of anion transporters consists of ten members that are found in various organs in the body including the stomach, intestine, kidney, thyroid and ear where they transport anions including bicarbonate, chloride and sulfate, typically in an exchange mode. Mutations in these genes cause a plethora of diseases such as diastrophic dysplasia affecting sulfate uptake into chondrocytes (SLC26A2), congenital chloride-losing diarrhoea (SLC26A3) affecting chloride secretion in the intestine and Pendred's syndrome (SLC26A4) resulting in hearing loss. To understand how these mutations affect the structures of the SLC26 membrane proteins and their ability to function properly, 12 human disease-causing mutants from SLC26A2, SLC26A3 and SLC26A4 were introduced into the equivalent sites of the sulfate transporter anti-sigma factor antagonist (STAS) domain of a bacterial homologue SLC26 protein DauA (YchM). Biophysical analyses including size-exclusion chromatography, circular dichroism (CD), differential scanning fluorimetry (DSF) and tryptophan fluorescence revealed that most mutations caused protein instability and aggregation. The mutation A463K, equivalent to N558K in human SLC26A4, which is located within α-helix 1 of the DauA STAS domain, stabilized the protein. CD measurements showed that most disease-related mutants had a mildly reduced helix content, but were more sensitive to thermal denaturation. Fluorescence spectroscopy showed that the mutants had more open structures and were more readily denatured by urea, whereas DSF indicated more labile folds. Overall, we conclude that the disease-associated mutations destabilized the STAS domain resulting in an increased propensity to misfold and aggregate.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Bai
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8.
authors.forename: Trevor F
authors.initials: TF
authors.lastname: Moraes
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8 r.reithmeier@utoronto.ca.
authors.forename: Reinhart A F
authors.initials: RA
authors.lastname: Reithmeier
mesh_terms: D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics; D027963:Chloride-Bicarbonate Antiporters / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D003967:Diarrhea / Q000151:congenital / Q000235:genetics; D004392:Dwarfism / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006044:Goiter, Nodular / Q000235:genetics; D006319:Hearing Loss, Sensorineural / Q000235:genetics; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000076988:Sulfate Transporters; D014357:Trypsin / Q000737:chemistry
chemical_list: D027321:Anion Transport Proteins; D027963:Chloride-Bicarbonate Antiporters; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C081011:SLC26A3 protein, human; C085609:SLC26A4 protein, human; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; C556152:YchM protein, E coli; D014357:Trypsin
keywords: SLC26; STAS domain; anion transporters; inherited diseases; membrane protein folding