The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding. Academic Article uri icon

Overview

abstract

  • Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following substrate binding. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. By contrast, an equilibrated structure does not show such classes. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties.

publication date

  • April 22, 2022

Research

keywords

  • Amino Acid Transport System X-AG
  • Archaea

Identity

PubMed Central ID

  • PMC9044058

Scopus Document Identifier

  • 85129778363

Digital Object Identifier (DOI)

  • 10.7554/eLife.42166

PubMed ID

  • 35452090

Additional Document Info

volume

  • 154

issue

  • 5