Search

In mammals, glucose transporters (GLUT) control organism-wide blood glucose homeostasis. In human, this is accomplished by fourteen different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarialplasmodium falciparumtransporterPfHT1, which is uniquely able to transport a wide range of different sugars.PfHT1 was captured in an intermediate “occluded” state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity inPfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed inPfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resemblesPfHT1. The coordination of fructose lowers the energetic barriers between the outward and inward-facing states, and the observed binding mode for fructose is consistent with biochemical analysis. Rather than a substrate binding site that achieves strict specificity by having a high-affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMS structure, and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 A. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 A from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics. Crystal structure of the Plasmodium falciparum hexose transporter PfHT1 reveals the molecular basis of its ability to transport multiple types of sugar as efficiently as the dedicated mammalian glucose and fructose transporters.

Significance Molecular mechanisms of general anesthetic modulation in pentameric ligand-gated ion channels remain controversial. Here we present molecular simulations and functional data that reveal correlations between dynamic differences in a membrane-accessible cavity and dramatic anesthetic effects, separate inhibitory and potentiating effects within the same electrophysiology recordings, and support a model for communication between the lipid bilayer and ion channel pore. In particular, enhanced electrostatic interactions in the membrane-accessible site were associated with a unique mode of anesthetic potentiation, persisting tens of minutes after washout. These results offer a bridge between lipid- and receptor-based theories of anesthesia, with the potential to inform both mechanistic understanding and drug development., Theories of general anesthesia have shifted in focus from bulk lipid effects to specific interactions with membrane proteins. Target receptors include several subtypes of pentameric ligand-gated ion channels; however, structures of physiologically relevant proteins in this family have yet to define anesthetic binding at high resolution. Recent cocrystal structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for the common surgical agent propofol (PFL), offering a detailed model system for anesthetic modulation. Here, we combine molecular dynamics and oocyte electrophysiology to reveal differential motion and modulation upon modification of a transmembrane binding site within each GLIC subunit. WT channels exhibited net inhibition by PFL, and a contraction of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in GLIC variants exhibiting net PFL potentiation, the cavity was persistently expanded and proximal to M2. Mutations designed to favor this deepened site enabled sensitivity even to subclinical concentrations of PFL, and a uniquely prolonged mode of potentiation evident up to ∼30 min after washout. Dependence of these prolonged effects on exposure time implicated the membrane as a reservoir for a lipid-accessible binding site. However, at the highest measured concentrations, potentiation appeared to be masked by an acute inhibitory effect, consistent with the presence of a discrete, water-accessible site of inhibition. These results support a multisite model of transmembrane allosteric modulation, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.

Theories of general anesthesia have shifted in focus from bulk lipid effects to specific interactions with membrane proteins. Target receptors include several subtypes of pentameric ligand-gated ion channels; however, structures of physiologically relevant proteins in this family have yet to define anesthetic binding at high resolution. Recent cocrystal structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for the common surgical agent propofol (PFL), offering a detailed model system for anesthetic modulation. Here, we combine molecular dynamics and oocyte electrophysiology to reveal differential motion and modulation upon modification of a transmembrane binding site within each GLIC subunit. WT channels exhibited net inhibition by PFL, and a contraction of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in GLIC variants exhibiting net PFL potentiation, the cavity was persistently expanded and proximal to M2. Mutations designed to favor this deepened site enabled sensitivity even to subclinical concentrations of PFL, and a uniquely prolonged mode of potentiation evident up to ∼30 min after washout. Dependence of these prolonged effects on exposure time implicated the membrane as a reservoir for a lipid-accessible binding site. However, at the highest measured concentrations, potentiation appeared to be masked by an acute inhibitory effect, consistent with the presence of a discrete, water-accessible site of inhibition. These results support a multisite model of transmembrane allosteric modulation, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.