Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Riederer, Erika A; Valiyaveetil, Francis I.

doi:10.1073/pnas.1907852116

“…However, this cooperativity is greatly suppressed when Met314 (Met311 in Glt Ph ) is replaced by Ala or Leu [37], as is the substrate affinity. Although no structures have been reported for any of these mutants, Trp fluorescence measurements show that the pattern of conformational changes in HP2 in response to ion and substrate binding [19,37] is also subtly altered upon mutation of Met314 [38,39].…”

Section: Introductionmentioning

confidence: 96%

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

Zhou

¹

,

Trinco

²

,

Slotboom

³

et al. 2021

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Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs GltPh and GltTk, revealed that this methionine is involved in the coordination of one of the three Na+ ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of GltPh showing that mutations of this methionine diminish the binding cooperativity between substrates and Na+. It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of GltTk using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism.

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“…Gating in the OFS, where only HP2 moves to bind Na + ions and L-asp is faster (Hanelt et al, 2015), although slow HP2 opening has also been proposed (Riederer and Valiyaveetil, 2019). Notably, kinetic studies showed that the release (and binding) of one Na + ion in the IFS, most likely Na2, is rapid (Oh and Boudker, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…These extensive conformational changes, involving repacking the domain interface, may explain why substrate gating is slow in the IFS ( Oh and Boudker, 2018 ). Gating in the OFS, where only HP2 moves to bind Na + ions and L-asp is faster ( Hänelt et al, 2015 ), although slow HP2 opening has also been proposed ( Riederer and Valiyaveetil, 2019 ). Notably, kinetic studies showed that the release (and binding) of one Na + ion in the IFS, most likely Na2, is rapid ( Oh and Boudker, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

“…As the transport domain translocates into the IFS, HP2 replaces HP1 on the domains interface, while HP1 now lines an intracellular vestibule leading to the substrate-binding site ( Figure 1—figure supplement 1 ). Structural and biophysical studies have established that HP2 serves as the transporter’s extracellular gate ( Boudker et al, 2007 ; Verdon et al, 2014 ; Focke et al, 2011 ; Riederer and Valiyaveetil, 2019 ). HP2 closes when the transporter is bound to Na + ions and L-asp and when it is empty ( Verdon et al, 2014 ; Yernool et al, 2004 ; Jensen et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

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

Wang

¹

,

Boudker

²

2020

eLife

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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.

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“…As the transport domain translocates into the IFS, HP2 replaces HP1 on the domains interface, while HP1 now lines an intracellular vestibule leading to the substrate-binding site (Figure 1 Supplementary Figure 1). Structural and biophysical studies have established that HP2 serves as the extracellular gate of the transporter (Boudker et al, 2007;Focke et al, 2011;Verdon et al, 2014;Riederer e Valiyaveetil, 2019). HP2 closes when the transporter is bound to Na + ions and L-asp and when it is empty (Yernool et al, 2004;Jensen et al, 2013;Verdon et al, 2014).…”

mentioning

confidence: 99%

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

Wang

¹

,

Boudker

²

2020

Preprint

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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. MAIN TEXTSodium and aspartate symporter GltPh is an archaeal homologue of human glutamate transporters, which clear the neurotransmitter glutamate from the synaptic cleft following rounds of neurotransmission (Danbolt, 2001). GltPh has served as a model system to uncover the structural and mechanistic features of glutamate transporters (Yernool et al., 2004;Boudker et al., 2007;Reyes et al., 2009;Akyuz et al., 2013;Erkens et al., 2013;Reyes et al., 2013;Verdon et al., 2014;Akyuz et al., 2015;Hanelt et al., 2015;Mcilwain et al., 2016;Scopelliti et al., 2018). Recently, structural studies on other members of the family, including human variants, have enriched the field and have been mostly consistent with earlier findings on GltPh (Canul-Tec et al., 2017;Garaeva et al., 2018;Yu et al., 2019).Collectively, these studies provide what appears to be a nearly complete picture of the structural changes that underlie transport. Briefly, the transporters are homotrimers with each protomer consisting of a centrally located scaffold or trimerization domain and a peripheral transport domain that harbors the L-aspartate (L-asp) and three sodium (Na + ) ions binding sites. The crucial conformational transition from the outward-facing state (OFS), in which L-asp binding site is near the extracellular solution, into the inward-facing

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Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Cited by 17 publications

References 43 publications

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

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

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

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