Heterogeneous substrate binding in a glutamate transporter homologue

Abstract

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family, including kinetic models of transport. Recent studies have revealed transport rate heterogeneity in an archaeal glutamate transporter homologue GltPh, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of GltPh, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type GltPh that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant GltPh provides multiple potential explanations of heterogeneous substrate binding. At equilibrium, we show subtle differences in tilts of protomers in the outward-facing state and configurations of the substrate-binding pocket. Within seconds of substrate binding, we observe perturbed helical packing of the extracellular half of the substrate-binding domain. Some or all of these may contribute to the heterogeneity observed in binding and transport.

An environmentally ultrasensitive fluorine probe to resolve protein conformational ensembles by19F NMR and cryo-EM

“…Motivated by this paradox, we imaged TBOA-bound RSMR by cryo-EM. Following particle alignment with imposed C3 symmetry, we performed symmetry expansion and 3D classification to sort multiple conformations of the transporter protomers 34, 37 . We observed 91 % protomers in IFS with a wide-open substrate-binding site occupied by TBOA ( Fig.…”

“…Intrigued by the observation that the dominant state of TBOA-bound RSMR mutant (inward-facing S2) differs from the dominant state of the WT transporter (outward-facing S4), we imaged it by cryo-EM. Following particle alignment with imposed C3 symmetry, we performed symmetry expansion and 3D classification without alignment to sort multiple conformations of the transporter protomers in the trimer 22,23 . We observed that the dominant conformation of the RSMR mutant is an IFS with a wide-open substrate-binding site occupied by TBOA ( Figure 2a , Extended Data Fig.…”

Distinct roles of the Na + binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, Glt Ph

“…Our data suggests that the binding of Asp to Glt Ph can take place through two different pathways. A recent study of a mutant D40N/S279E and the wild-type Glt Ph transporter has also reported heterogenous Asp binding consistent with two binding modes for Asp ( 32 ). The conventional pathway consists of Na + binding to the Na1 and the Na3 sites prior to Asp binding.…”

Distinct roles of the Na+ binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, GltPh

“…A recent study of a mutant D40N/S279E and the wild GltPh transporter has also reported heterogenous Asp binding consistent with two binding modes for Asp. (32) The conventional pathway consists of Na + binding to the Na1 and the Na3 sites prior to Asp binding. In the alternate pathway, the binding of Asp takes place after the initial binding of Na + to the Na1 site.…”