Structural basis of ion – substrate coupling in the Na⁺-dependent dicarboxylate transporter VcINDY

Abstract

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen induced-fit mechanism. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's induced-fit mechanism is a result of the sodium-dependent formation of the substrate binding site.

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

“…That the VcINDY conformation is effectively independent of dicarboxylate identity suggests that this fully bound conformation is very stable—as predicted in our modeling of optimal co-substrate binding. Moreover, recent PEG-modification protection experiments demonstrated that, in the presence of high concentrations of only Na + , residues associated with the substrate binding pocket are protected from PEG modification (Sampson et al, 2020; Sauer et al, 2022a). This suggests some interaction between Na + and the apo VcINDY binding pocket, which is consistent with a Na + -first binding order and with the prediction that slippage is minimized and transport is optimized by weak binding of the first co-substrate(s) (Fig.…”

“…1A) (Mulligan et al, 2014;Fitzgerald et al, 2017). Over the past decade, extensive in vitro biochemical characterization (Mulligan et al, 2014(Mulligan et al, , 2016Fitzgerald et al, 2017;Sampson et al, 2020Sampson et al, , 2021, and extensive structural insight from Xray crystallography, cryo-electron microscopy (cryo-EM), and computational studies (Mancusso et al, 2012;Mulligan et al, 2016;Nie et al, 2017;Sauer et al, 2021Sauer et al, , 2022a, have made VcINDY into the prototypical transporter for understanding the mechanistic details of transport in the DASS family.…”