Structure and function of the divalent anion/Na+ symporter from Vibrio cholerae and a humanized variant

Abstract

Integral membrane proteins of the divalent anion/Na+ symporter (DASS) family translocate dicarboxylate, tricarboxylate or sulphate across cell membranes, typically by utilizing the preexisting Na+ gradient. The molecular determinants for substrate recognition by DASS remain obscure, largely owing to the absence of any substrate-bound DASS structure. Here we present 2.8-Å resolution X-ray structures of VcINDY, a DASS from Vibrio cholerae that catalyses the co-transport of Na+ and succinate. These structures portray the Na+-bound VcINDY in complexes with succinate and citrate, elucidating the binding sites for substrate and two Na+ ions. Furthermore, we report the structures of a humanized variant of VcINDY in complexes with succinate and citrate, which predict how a human citrate-transporting DASS may interact with its bound substrate. Our findings provide insights into metabolite transport by DASS, establishing a molecular basis for future studies on the regulation of this transport process.

Substrate-dependent accessibility changes in the Na+-dependent C4-dicarboxylate transporter, VcINDY, suggest differential substrate effects in a multistep mechanism

“…In both VcINDY and GltPh, the tips of these re-entrant loops form part of the substrate binding site, coordinating both the coupling ion and substrate 18,19,42 . However, a major mechanistic difference between VcINDY and GltPh is the role of the re-entrant loops in gating and coupling.…”

“…However, a major mechanistic difference between VcINDY and GltPh is the role of the re-entrant loops in gating and coupling. In GltPh and other glutamate transport homologues, the substrate and coupling ions are fully enclosed in the transport domain 23,[42][43][44] In the IFS structure and OFS model of VcINDY, the substrate is solvent exposed and straddles the interface of the scaffold and transport domains 18,19,22 , potentially precluding the need for hairpincoordinated gating in DASS transporters. In this study, we have probed the substrate-dependent accessibility of several residues in the re-entrant hairpin loops of VcINDY; M157 and T154 in HP1, and S381, L384, and V388 in HP2 (Fig.…”

“…The DASS transporter family belongs to the Ion Transporter (IT) Superfamily 10,11 , and the majority of characterised DASS transporters are known to transport their anionic substrates coupled to the co-transport of multiple Na + ions 12 13-17 . All of our current structural and mechanistic understanding of the DASS family comes from studies on a bacterial family member, VcINDY, from Vibrio cholerae, which is the only DASS transporter for which we have high resolution structural information 18,19 .…”

“…The x-ray structures of VcINDY reveal that it forms a homodimer and each protomer consists of two domains; the scaffold domain that forms dimer interface contacts, and the transport domain that houses key substrate binding residue ( Fig. 1A) 18,19 . All structures of VcINDY to date are captured in the same IFS conformation where the substrate is exposed to the cytoplasmic side of the membrane suggesting that the crystallisation conditions select for this lowest free-energy state conformation of VcINDY 18,19 .…”

“…1A) 18,19 . All structures of VcINDY to date are captured in the same IFS conformation where the substrate is exposed to the cytoplasmic side of the membrane suggesting that the crystallisation conditions select for this lowest free-energy state conformation of VcINDY 18,19 . Using a combination of symmetry-based structural modelling and extensive biochemical and biophysical validation, we have described an OFS conformation of VcINDY in which the transport domain and the substrate binding site are vertically translocated through the membrane (Fig.…”