Structure and mechanism of the K+/H+ exchanger KefC.

Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, K+ levels are maintained by various ion transporters. One major family is the proton-driven K+ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K+ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na+/H+ antiporter NapA. By structural assignment of a coordinated K+ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K+ ion. KefC harbors C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K+-ion-channels.

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5.

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 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 malarial Plasmodium falciparum transporter PfHT1, 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 in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 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 resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-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.

Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning.

Sugar porters (SPs) represent the largest group of secondary-active transporters. Some members, such as the glucose transporters (GLUTs), are well known for their role in maintaining blood glucose homeostasis in mammals, with their expression upregulated in many types of cancers. Because only a few sugar porter structures have been determined, mechanistic models have been constructed by piecing together structural states of distantly related proteins. Current GLUT transport models are predominantly descriptive and oversimplified. Here, we have combined coevolution analysis and comparative modeling, to predict structures of the entire sugar porter superfamily in each state of the transport cycle. We have analyzed the state-specific contacts inferred from coevolving residue pairs and shown how this information can be used to rapidly generate free-energy landscapes consistent with experimental estimates, as illustrated here for the mammalian fructose transporter GLUT5. By comparing many different sugar porter models and scrutinizing their sequence, we have been able to define the molecular determinants of the transport cycle, which are conserved throughout the sugar porter superfamily. We have also been able to highlight differences leading to the emergence of proton-coupling, validating, and extending the previously proposed latch mechanism. Our computational approach is transferable to any transporter, and to other protein families in general.

Molecular Basis of Coupled Transport and Anion Conduction in Excitatory Amino Acid Transporters.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1-5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na+ ions and one H+ in exchange with one K+ to accumulate glutamate against up to 106-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na+- or H+-coupled prokaryotic transporters and Na+-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions.

g_elpot: A Tool for Quantifying Biomolecular Electrostatics from Molecular Dynamics Trajectories.

Electrostatic forces drive a wide variety of biomolecular processes by defining the energetics of the interaction between biomolecules and charged substances. Molecular dynamics (MD) simulations provide trajectories that contain ensembles of structural configurations sampled by biomolecules and their environment. Although this information can be used for high-resolution characterization of biomolecular electrostatics, it has not yet been possible to calculate electrostatic potentials from MD trajectories in a way allowing for quantitative connection to energetics. Here, we present g_elpot, a GROMACS-based tool that utilizes the smooth particle mesh Ewald method to quantify the electrostatics of biomolecules by calculating potential within water molecules that are explicitly present in biomolecular MD simulations. g_elpot can extract the global distribution of the electrostatic potential from MD trajectories and measure its time course in functionally important regions of a biomolecule. To demonstrate that g_elpot can be used to gain biophysical insights into various biomolecular processes, we applied the tool to MD trajectories of the P2X3 receptor, TMEM16 lipid scramblases, the secondary-active transporter GltPh, and DNA complexed with cationic polymers. Our results indicate that g_elpot is well suited for quantifying electrostatics in biomolecular systems to provide a deeper understanding of its role in biomolecular processes.

Allosteric gate modulation confers K+ coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) mediate glial and neuronal glutamate uptake to terminate synaptic transmission and to ensure low resting glutamate concentrations. Effective glutamate uptake is achieved by cotransport with 3 Na+ and 1 H+ , in exchange with 1 K+ . The underlying principles of this complex transport stoichiometry remain poorly understood. We use molecular dynamics simulations and electrophysiological experiments to elucidate how mammalian EAATs harness K+ gradients, unlike their K+ -independent prokaryotic homologues. Glutamate transport is achieved via elevator-like translocation of the transport domain. In EAATs, glutamate-free re-translocation is prevented by an external gate remaining open until K+  binding closes and locks the gate. Prokaryotic GltPh contains the same K+ -binding site, but the gate can close without K+ . Our study provides a comprehensive description of K+ -dependent glutamate transport and reveals a hitherto unknown allosteric coupling mechanism that permits adaptions of the transport stoichiometry without affecting ion or substrate binding.

Gating Charge Calculations by Computational Electrophysiology Simulations.

Electrical cell signaling requires adjustment of ion channel, receptor, or transporter function in response to changes in membrane potential. For the majority of such membrane proteins, the molecular details of voltage sensing remain insufficiently understood. Here, we present a molecular dynamics simulation-based method to determine the underlying charge movement across the membrane-the gating charge-by measuring electrical capacitor properties of membrane-embedded proteins. We illustrate the approach by calculating the charge transfer upon membrane insertion of the HIV gp41 fusion peptide, and validate the method on two prototypical voltage-dependent proteins, the Kv1.2 K+ channel and the voltage sensor of the Ciona intestinalis voltage-sensitive phosphatase, against experimental data. We then use the gating charge analysis to study how the T1 domain modifies voltage sensing in Kv1.2 channels and to investigate the voltage dependence of the initial binding of two Na+ ions in Na+-coupled glutamate transporters. Our simulation approach quantifies various mechanisms of voltage sensing, enables direct comparison with experiments, and supports mechanistic interpretation of voltage sensitivity by fractional amino acid contributions.