The mechanism of sodium and substrate release from the binding pocket of vSGLT

Abstract

Membrane co-transport proteins that utilize a 5-helix inverted repeat motif have recently emerged as one of the largest structural class of secondary active transporters1,2. However, despite many structural advances there is no clear evidence as to how ion and substrate transport are coupled. Here, we report a comprehensive study of the Sodium-Galactose Transporter from Vibrio parahaemolyticus (vSGLT) consisting of molecular dynamics simulations, biochemical characterization, and a new crystal structure of the inward-open conformation at 2.7 Å resolution. Our data show that sodium exit causes a reorientation of transmembrane helix 1 (TM1) opening an inner gate required for substrate exit, while also triggering minor rigid body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose to readily enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium binding sites, the unwound portion of TM1, and the substrate binding site that is essential in the coupling of co-transport.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…Binding to this site is affected by mutagenesis of S365 in vSGLT to alanine and of analogous residues in other SSS proteins (29)(30)(31). Consistent with the observations for Mhp1, MD simulations of these inward-facing conformations of vSGLT show that ions are released quickly from the proposed binding site (28,(32)(33)(34)(35).…”

“…S2B) and with the absence of densities at these sites in structures of other states (7). Nevertheless, the simulation results could be attributed to the fact that the structure used has an inward-facing conformation to which ion binding appears to be weak for other FIRL transporters (11,17,28,(32)(33)(34). Therefore we also measured the apparent sodium affinity of BetP after sitedirected mutagenesis of the side chains proposed to coordinate the ions directly, i.e., S306 and M310 from pNa2 ( Table S1).…”

“…In structures of vSGLT, which also are inward facing, no suitable densities for sodium ions have been observed (13,28), although a site was proposed by analogy to Na2 in LeuT (Fig. 1).…”

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

“…Typically, transporter crystal structures are classified as inward-facing, outwardfacing, or occluded on the basis of the accessibility of the substrate binding site (7)(8)(9)(10)(11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13).…”

“…Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7)(8)(9)(10)(11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4,7,(9)(10)(11)(12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outwardfacing, or occluded on the basis of the accessibility of the substrate binding site (7)(8)(9)(10)(11).…”