Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-Coupled Transporter Extracellular Topography [Membrane Biology]

January 7th, 2015 by Zhu, Q., Kao, L., Azimov, R., Abuladze, N., Newman, D., Kurtz, I.

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 structure-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 2 intramolecular disulfide bonds, Cys583-585 and Cys617-642 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.