title: Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.
abstract: The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.
authors.affiliation: From the Department of Medicine and quzhu@mednet.ucla.edu.
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: From the Department of Medicine and.
authors.forename: Liyo
authors.initials: L
authors.lastname: Kao
authors.affiliation: From the Department of Medicine and.
authors.forename: Rustam
authors.initials: R
authors.lastname: Azimov
authors.affiliation: From the Department of Medicine and.
authors.forename: Natalia
authors.initials: N
authors.lastname: Abuladze
authors.affiliation: From the Department of Medicine and.
authors.forename: Debra
authors.initials: D
authors.lastname: Newman
authors.affiliation: From the Department of Medicine and Brain Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D017136:Ion Transport / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D003545:Cysteine
keywords: Disulfide; Glycosylation; Membrane Protein; Protein Folding; Transporter