Biology subjects

Boudker, O.

Publications and source records attributed to Boudker, O..

11 recordsLinked to original sources

bioRxiv2026

Multimodal dynamics control activity of a glial glutamate transporter

Membrane transporters move polar solutes across lipid bilayers to regulate cellular metabolism, signaling, and drug distribution. These proteins operate via an alternating-access mechanism, cycling between extracellular-, intermediate-, and intracellular-facing conformations. The human excitatory amino acid transporter 1 (EAAT1) protects neurons from excitotoxic damage by mediating the uptake of glutamate and aspartate into glial cells. Defects in EAAT1 function result in numerous pathologies, including epilepsy and ataxia, suggesting that positive modulation of these transporters might ameliorate glutamate neurotoxicity. However, developing EAAT1 activators requires understanding the timing of conformational changes, which remain largely unexplored. Here, we establish an experimental platform that combines single-molecule Forster resonance energy transfer (smFRET) to monitor real-time conformational dynamics, single-transporter activity assays to correlate dynamics with function, and cryogenic electron microscopy (cryoEM) to visualize discrete conformations at high resolution. This platform enables detection of [A]ngstrom-scale movements of single transporter molecules in real time, revealing that EAAT1 intersperses rapid conformational dynamics with long pauses. Slow and fast dynamics can be modulated by substrates, membrane composition, and mutations, and are correlated with the enrichment of specific structural states. We leverage this platform to investigate an EAAT1 mutation associated with severe episodic ataxia and show that it inhibits transport by stabilizing a paused cytoplasm-facing conformation. These results identify multimodal dynamics as an intrinsic, regulatable feature of EAAT1 function and, therefore, a potential therapeutic target. Henceforth, our integrated platform will facilitate investigations of other regulatory factors, including the effects of small-molecule and lipid modulators on the transport cycle.

biophysics↗

bioRxiv2025

Mechanism and Structure-Guided Optimization of SLC1A1/EAAT3-Selective Inhibitors in Kidney Cancer

Renal Cell Carcinomas (RCCs) depend metabolically on the trimeric sodium-coupled aspartate and glutamate transporter, SLC1A1/EAAT3; however, pharmacologically targeting SLC1A1 is challenging. We determined a cryo-EM structure of human SLC1A1 bound to compound 3e, a recently described SLC1A1-selective bicyclic imidazo[1,211]pyridine-3-amine (BIA) inhibitor. 3e binds a membrane-embedded allosteric pocket accessible only in the apo state, when SLC1A1 is unbound to substrate and sodium, and likely prevents sodium and substrate binding. Moreover, by wedging between the trimerization domain and the substrate-binding transport domain, alongside a cholesterol moiety from the lipid bilayer, 3e blocks SLC1A1s elevator-like movements that are essential for transport. Mutations in this binding pocket abolish 3e interaction and counteract 3es cytotoxicity in RCC cells, confirming on-target activity and explaining SLC1A1 selectivity. A structure-guided medicinal chemistry effort yielded two new SLC1A1-selective BIA derivatives, PBJ1 and PBJ2, with enhanced cytotoxicity resulting from the inhibition of SLC1A1-dependent aspartate, glutamate, and cysteine metabolism.

cancer biology↗

bioRxiv2024

Structural basis of the excitatory amino acid transporter 3 substrate recognition

Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.

biophysics↗

bioRxiv2023

Uncoupled substrate binding underlies the evolutionary switch between Na+ and H+-coupled prokaryotic aspartate transporters

Secondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to changing environments and needs. The bases of such diversification, and thus principles of ion coupling, are unexplored. Employing phylogenetics and ancestral protein reconstruction, we investigated sodium-coupled transport in prokaryotic glutamate transporters, a mechanism ubiquitous across life domains and critical to neurotransmitter recycling in humans. We found that the evolutionary transition from sodium-dependent to independent substrate binding to the transporter preceded changes in the coupling mechanism. Structural and functional experiments suggest that the transition entailed allosteric mutations, making sodium binding dispensable without affecting ion-binding sites. Allosteric tuning of transporters energy landscapes might be a widespread route of their functional diversification.

biochemistry↗

bioRxiv2022

Dancing with the ions: symport and antiport mechanisms of human glutamate transporters

Excitatory amino acid transporters (EAATs) pump glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton and counter-transporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms remain unclear. We report high-resolution Cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or in the absence of ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the counter-transported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.

biophysics↗

bioRxiv2022

An environmentally ultrasensitive fluorine-19 NMR probe for monitoring protein conformational equilibria

Limited chemical shift dispersion represents a significant barrier to studying multi-state equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in 1D NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy. Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and 3D classification.

biophysics↗

bioRxiv2021

Heterogeneous substrate binding in a glutamate transporter homologue

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family. Recent studies revealed transport rate heterogeneity in an archaeal homologue GltPh, inconsistent with simple kinetic models, but its structural and mechanistic determinants remain undefined. In a mutant GltPh, which exclusively populates the outward-facing state, we demonstrate the co-existence of at least two sub-states in slow equilibrium binding the substrate with different apparent affinities. Wild-type GltPh shows similar binding properties, and modulation of the sub-state equilibrium correlates with transport rates. Following binding, the low-affinity sub-state of the mutant is transient. 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. In contrast, an equilibrated structure does not show such classes. The structure at 2.2 [A] 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, while the extracellular labile half sets the affinity and dynamic properties.

biophysics↗

bioRxiv2020

Transport mechanism of the neuronal excitatory amino acid transporter

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report Cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing for the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which conserved glutamate mediates proton symport.

biophysics↗

bioRxiv2020

Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homologue, sodium and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.

biophysics↗

bioRxiv2020

The high-energy transition state of a membrane transporter

Membrane transporters mediate cellular uptake of nutrients, signaling molecules and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of a model glutamate transporter homologue suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate-limiting to transport. Here, we aim to gain insight into the structure of the high-energy transition state that limits the rate of this critical isomerization reaction. Using bioinformatics, we identify gain-of-function mutants of the transporter and apply linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach for allosteric modulation of these transporters.

biophysics↗

bioRxiv2019

Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

biophysics↗