David M. MacLean

Ancestral reconstruction of the ligand-binding pocket of Family C G protein-coupled receptors

The metabotropic glutamate receptors (mGluRs) within the Family C subclass of G protein-coupled receptors are crucial modulators of synaptic transmission. However, their closest relatives include a diverse group of sensory receptors whose biological functions are not associated with neurotransmission, raising the question of the evolutionary origin of amino acid-binding Family C receptors. A common feature of most, if not all, functional Family C receptors is the presence of an amino acid-binding site localized within the large extracellular Venus flytrap domain. Here, we used maximum likelihood methods to infer the ancestral state of key residues in the amino acid-binding pocket of a primordial Family C receptor. These residues were reconstructed in the background of the fish 5.24 chemosensory receptor, a broad-spectrum amino acid-activated receptor. Unlike the WT 5.24 receptor, which was not activated by mGluR agonists and displayed low sensitivity toward l-glutamate, the reconstructed ancestral receptor possessed a pharmacological profile characterized by high affinity for both l-glutamate and selective Group I mGluR agonists. This pharmacological phenotype could be largely recapitulated by mutating only two residues in the 5.24 receptor-binding pocket. Our results suggest that this primordial Family C receptor may have arisen early in metazoan evolution and that it already was preadapted as a glutamate receptor for its later use at excitatory synapses in glutamate-mediated neurotransmission.

Dynamic membrane protein topological switching upon changes in phospholipid environment

Significance Understanding how a protein sequence folds and orients in a lipid bilayer is central to establishing the molecular basis for membrane protein organization. How lipid environment affects membrane protein organization is understudied. We established that membrane protein orientation is dynamic during and after assembly, dependent on membrane lipid composition, and independent of other cellular factors. We developed a proteoliposome system in which lipid composition can be controlled before and after membrane protein reconstitution and used it to assess the kinetics of changes in transmembrane domain (TMD) orientation and phospholipid flipping within the lipid bilayer triggered by a change in lipid composition. We demonstrate that membrane proteins can undergo rapid postassembly TMD flipping in response to changes in the lipid environment. A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

Background: Agonist binds to an extracellular agonist-binding domain in AMPA receptors. Results: Willardiines induce a range of cleft closure states in the agonist-binding domain of AMPA receptors. Conclusion: The fraction of the agonist-binding domains in a closed cleft conformation correlates with the extent of activation. Significance: The dynamics and extent of cleft closure in the agonist-binding domain control activation of AMPA receptors. We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the “closed” cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.

Amino-terminal Domain Tetramer Organization and Structural Effects of Zinc Binding in the N-Methyl-d-aspartate (NMDA) Receptor

Background: NMDA receptors are ion channels activated by glutamate and glycine and inhibited by zinc. Results: Zinc binding causes a decrease in distance between the amino-terminal domain lower and upper lobes without affecting intersubunit distances. Conclusion: Zinc induces cleft closure in the amino-terminal domain without causing large scale rearrangements in the upper lobe of the ATD tetramer. Significance: This work demonstrates zinc-induced conformational changes in a functional NMDA receptor. N-Methyl-d-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian central nervous system. An important feature of these receptors is their capacity for allosteric regulation by small molecules, such as zinc, which bind to their amino-terminal domain (ATD). Zinc inhibition through high affinity binding to the ATD has been examined through functional studies; however, there is no direct measurement of associated conformational changes. We used luminescence resonance energy transfer to show that the ATDs undergo a cleft closure-like conformational change upon binding zinc, but no changes are observed in intersubunit distances. Furthermore, we find that the ATDs are more closely packed than the related AMPA receptors. These results suggest that the stability of the upper lobe contacts between ATDs allow for the efficient propagation of the cleft closure conformational change toward the ligand-binding domain and transmembrane segments, ultimately inhibiting the channel.

Na+/Cl− Dipole Couples Agonist Binding to Kainate Receptor Activation

Kainate-selective ionotropic glutamate receptors (GluRs) require external Na+ and Cl− as well as the neurotransmitter l-glutamate for activation. Although, external anions and cations apparently coactivate kainate receptors (KARs) in an identical manner, it has yet to be established how ions of opposite charge achieve this. An additional complication is that KARs are subject to other forms of cation modulation via extracellular acidification (i.e., protons) and divalent ions. Consequently, other cation species may compete with Na+ to regulate the time KARs remain in the open state. Here we designed experiments to unravel how external ions regulate GluR6 KARs. We show that GluR6 kinetics are unaffected by alterations in physiological pH but that divalent and alkali metal ions compete to determine the time course of KAR channel activity. Additionally, Na+ and Cl− ions coactivate GluR6 receptors by establishing a dipole, accounting for their common effect on KARs. Using charged amino acids as tethered ions, we further demonstrate that the docking order is fixed with cations binding first, followed by anions. Together, our findings identify the dipole as a novel gating feature that couples neurotransmitter binding to KAR activation.

Stargazin promotes closure of the AMPA receptor ligand-binding domain

Stargazin enhances closure of the AMPA receptor ligand-binding domain, thereby facilitating channel activation.

Subtype-dependent N-Methyl-d-aspartate Receptor Amino-terminal Domain Conformations and Modulation by Spermine

Background: Amino-terminal domains (ATDs) of NMDA receptors dictate open probability and bind the modulator spermine. Results: GluN2A ATD is more open than GluN2B; GluN1 ATD is more open in the presence of GluN2A, and spermine binding opens the ATDs of GluN1 and GluN2B. Conclusion: The ATD conformations correlate to the open probability. Significance: ATD conformations depend on the subunit composition and change upon modulator binding. The N-methyl-d-aspartate (NMDA) subtype of the ionotropic glutamate receptors is the primary mediator of calcium-permeable excitatory neurotransmission in the central nervous system. Subunit composition and binding of allosteric modulators to the amino-terminal domain determine the open probability of the channel. By using luminescence resonance energy transfer with functional receptors expressed in CHO cells, we show that the cleft of the amino-terminal domain of the GluN2B subunit, which has a lower channel open probability, is on average more closed than the GluN2A subunit, which has a higher open probability. Furthermore, the GluN1 amino-terminal domain adopts a more open conformation when coassembled with GluN2A than with GluN2B. Binding of spermine, an allosteric potentiator, opens the amino-terminal domain cleft of both the GluN2B subunit and the adjacent GluN1 subunit. These studies provide direct structural evidence that the inherent conformations of the amino-terminal domains vary based on the subunit and match the reported open probabilities for the receptor.

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

Non‐Technical Summary  Communication between neurons is often carried out by neurotransmitters, such as glutamate, and their receptor proteins, such as AMPA‐type glutamate receptors. It has become clear that these AMPA receptors are not alone in cell membranes but are often associated with auxiliary proteins which alter their responsiveness to blocking drugs. In particular, the transmembrane AMPA receptor regulatory protein (TARP) family of auxiliary proteins has been argued to make the receptor less sensitive to antagonists and more sensitive to neurotransmitter. Here we apply basic pharmacological principles to argue that these two effects are not separate but linked to each other, i.e. AMPA receptors are less sensitive to antagonists because they are more sensitive to neurotransmitter. We further highlight that when considering the very rapid nature of signalling between nerve cells, neurotransmitters have insufficient time to dislodge antagonists from their binding site. As a result, antagonists appear to work through a different mechanism.

Proton-mediated conformational changes in an acid-sensing ion channel

Background: Protons activate acid-sensing ion channels (ASICs). Results: Proton binding leads to a movement involving the thumb and finger subdomains. Mutation of carboxylates lining the finger leads to loss of activation and loss of this movement. Conclusion: The carboxylates lining the finger domain are essential for the movement of the thumb and finger domains and in activation. Significance: This study provides insight into proton-induced conformational changes in ASICs. Acid-sensing ion channels are cation channels activated by external protons and play roles in nociception, synaptic transmission, and the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding, there is a conformational change that increases LRET efficiency between the probes at the thumb and finger subdomains in the extracellular domain of acid-sensing ion channels. Additionally, we show that this conformational change is lost upon mutating Asp-238, Glu-239, and Asp-260, which line the finger domains, to alanines. Electrophysiological studies showed that the single mutant D260A shifted the EC50 by 0.2 pH units, the double mutant D238A/E239A shifted the EC50 by 2.5 pH units, and the triple mutant D238A/E239A/D260A exhibited no response to protons despite surface expression. The LRET experiments on D238A/E239A/D260A showed no changes in LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which are involved in carboxyl/carboxylate interactions, are essential for proton-induced conformational changes in the extracellular domain, which in turn are necessary for receptor activation.

A conserved structural mechanism of NMDA receptor inhibition: A comparison of ifenprodil and zinc

Zinc and ifenprodil induce similar conformational changes in the NMDA receptor, suggesting a conserved mechanism of inhibition independent of receptor subtype and the site at which the inhibitor binds.