CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1

Abstract

Na+-coupled acid–base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid–base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

Structural basis for functional interactions in dimers of SLC26 transporters

“…The model of the SLC26Dg dimer displays a protomer-protomer membrane interface that is remarkably different from the membrane interfaces observed for the SLC4 and SLC23 families, both in its location and in its size 17,18,19,21,22 . Whereas the membrane dimer interfaces of SLC4 and SLC23 proteins center around TM6, and TM5 plus TM12, respectively, the midpoint of the SLC26Dg dimer is TM14 on the opposite side of the gate domain.…”

“…Dimeric states have been previously observed for pro-and eukaryotic members of the SLC4 25,26,27 , SLC23 28 , and SLC26 4,29,30,31,32 families. Recent structures subsequently confirmed this oligomeric state for SLC4 17,18,19 and SLC23 21,22 proteins and indicated that in both families the gate domains form the main interaction surface between protomers, though each family appears to hold a distinct dimer interface. As the crystal structure of SLC26Dg captured the protein in a monomeric state, the mode of interaction between SLC26 protomers has remained elusive.…”

“…Since SLC26Dg is monomeric in its detergent-solubilized state, we reconstituted spin-labeled protomers in lipid membranes to assure dimer formation 4 . As the gate domain in the SLC4 17,18,19 and SLC23 21,22 families establishes the main protomer-protomer contacts in the membrane (Fig. 1a,b), we engineered spin-labels at 13 different positions of the termini of gate domain helices in SLC26Dg.…”

“…The SLC26Dg membrane domain holds two intertwined inverted repeats of seven transmembrane segments (TMs). Despite a poor sequence homology, the SLC26 family shares this 7-TM inverted repeat (7TMIR) architecture with the SLC4 and SLC23 families that transport bicarbonate and nucleobases plus vitamin C, respectively 15,16,17,18,19,20,21,22 . The fourteen TMs are arranged in two subdomains: a compact core domain that holds the substrate binding site as inferred from the location of the nucleobases in the SLC23 crystal structures 20,21,22 , and an elongated gate domain that shields one side of the core domain.…”

Exploring the autoinhibitory domain of the electrogenic Na+/HCO3− transporter NBCe1‐B, from residues 28 to 62

“…Recently, Huynh et al . (2018) determined the structure at 3.9 Å of the membrane‐domain dimer of human NBCe1 by cryo‐electron microscopy (cryo‐EM). However, they could not model the cytosolic Nt, part of extracellular loop 3 (EL3), EL4, intracellular loop 5 (IL5), and cytosolic Ct due to the dynamic nature of these regions, which nevertheless could be very important for protein regulation.…”