The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding

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.

Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter

“…The deviation from the 1:1 model indicates a complex binding behavior, which is maybe not surprising considering that at least two separate binding interfaces (stator/N-lobe and elevator/C-lobe) are involved, combined with the conformational flexibility of the participating molecules. Similar observations of complex binding events in transport mechanisms have been made with the BtuCD-BtuF ABC transporter 65 or the GltPh elevator-type transporter 66 . Clearly, more detailed experiments are needed to fully understand the binding kinetics of the tripartite complex.…”

Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by 19F NMR and Cryo-EM

“…To understand the structural origin of this state, we imaged TBOA-bound RSMR by cryo-EM. Following particle alignment with imposed C3 symmetry, we performed symmetry expansion and three-dimensional (3D) classification to sort multiple conformations of the transporter protomers. , We observed 91% protomers in the IFS with a wide-open substrate-binding site occupied by TBOA (Figures b and S6, and Table S1; see the Materials and Methods Section for data processing details). The remaining protomers were in the OFS, similar to the TBOA-bound WT (RMSD of 0.646 Å).…”

Evolutionary analysis reveals the origin of sodium coupling in glutamate transporters

“…Introducing these and similar mutations in GltPh can reduce Na + binding and diminish coupling in some cases (10,(36)(37)(38). Though the proton carrier residues of GT-H are unknown, we previously suggested that a glutamate residue at the GltPh S279 position at the tip of HP1, conserved in GT-H, is a candidate (36). These sequence changes do not explain how a high-affinity substrate-binding site forms in a Na + -independent manner in GT-H and whether substrate binding to GT-H is H + -dependent or H + -coupling occurs through a different mechanism.…”

“…Thus, coupled binding and transport occur because the high-affinity substrate binding state is a high-energy state only accessible when Na + binds. Accordingly, mutations in all three Na + -binding sites diminish coupled substrate binding and transport (3,6,(33)(34)(35)(36).…”