SLC26Dg Crystal Structure
The first reported structure of a full-length SLC26/SulP polypeptide, including both TMD and cytoplasmic STAS domain, was from SLC26 of Deinoccocus geotherma- lis (SLC26Dg) (Geertsma et al. 2015). The SLC26Dg TMD shares 46 % and 57 % amino acid sequence similarity with the corresponding regions of human prestin and of E. coli DauA. Initial crystallization attempts achieved a structural resolution of only 7.2 A. Co-crystallization with an immunospecific anti-SLC26Dg nanobody allowed structural resolution of 3.2 A, without alteration of the TMD orientation of SLC26Dg.
The SLC26Dg structure reveals a UraA fold similar to that previously predicted and used as a modeling template for rat and chicken prestin/SLC26A5 (Gorbunov et al. 2014) and for human AE1/SLC4A1 (Barneaud-Rocca et al. 2013). Indeed, the X-ray crystal structure of human AE1 ultimately confirmed its UraA fold (Arakawa et al. 2015). The UraA fold shared by SLC26Dg and by AE1/SLC4A1 consists of 14 TMD a-helices of variable length arranged as two inverted repeats, each containing 7 a-helices. Pseudosymmetry-related helices 1-4 and 8-11 form a “core domain,” whereas helices 5-7 and 12-14 from an elongated “gate domain” that shields the core domain. Each inverted repeat includes one “broken” transmembrane span (spans 3 and 10) comprising a short a helix joined by flexible linker to a short p-sheet oriented at divergent angles within the bilayer, and contributing to a proposed anion substrate-binding site (no ligand was detected in the SLC26Dg crystals). The SLC26Dg crystal structure revealed the polypeptide in its inwardfacing conformation, exposing the substrate anion-binding site to the cytosol, and occluding it from the extracellular medium.
Purified SLC26Dg was monomeric in dodecyl maltoside but reconstituted into proteoliposomes as a dimer, consistent with previous evidence of stable dimers in solution, based on pulsed electron-electron double resonance spectroscopy and small-angle neutron scattering experiments (Compton et al. 2014; Compton et al. 2011). TMD monomeric dimensions of 45 A x 60 A x 40 A suggest minimal protrusion of the polypeptide beyond either surface of the lipid bilayer (Geertsma et al. 2015). Each monomer is believed to harbor an independent anion translocation pathway. However, one monomer can influence its dimeric partner’s conformation as also suggested by prestin, for which co-expression of two mutant polypeptides of distinct voltage-dependences yielded a unique electrical signature, consistent with functional inter-protomer interaction within the oligomer (Detro-Dassen et al. 2008). Regulated modulation of SLC26 oligomeric state has been postulated, but not yet reported.