Chunfeng Zhao

The Role of Local Hydration and Hydrogen-Bonding Dynamics in Ion and Solute Release from Ion-Coupled Secondary Transporters

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.

The molecular mechanism of ion-dependent gating in secondary transporters.

LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method) followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5). The Potential of Mean Force (PMF) computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrates binds to the site S1 ∼5 kcal/mol more favorable than that to the site S2 for all studied bound combinations of ions and a substrate.

Sodium channel selectivity and conduction: Prokaryotes have devised their own molecular strategy

The molecular strategy for alkali cation selectivity by a bacterial sodium channel resembles those of eukaryotic calcium and potassium channels, rather than those of eukaryotic sodium channels.

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters

Background: The intramolecular pathways propagating the impact of Na+ binding in neurotransmitter:sodium symporters (NSSs) are not sufficiently understood. Results: We identified computationally and verified experimentally an interaction network connecting Na+ binding with the intracellular gate. Conclusion: The identified pathways are conserved between bacterial LeuT and eukaryotic hDAT. Significance: We gain a new understanding of the structural basis for the functional role of Na+ binding in NSSs. Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na+-dependent reuptake of released neurotransmitters. Previous studies suggested that Na+-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na+ binding and transport (i.e. replacing Na+ with Li+ or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na+ cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na+ dependence. Thus, the detailed AIN generated from our method is shown to connect Na+ binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na+ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.

Evaluations of the Absolute and Relative Free Energies for Antidepressant Binding to the Amino Acid Membrane Transporter LeuT with Free Energy Simulations

The binding of ligands to protein receptors with high affinity and specificity is central to many cellular processes. The quest for the development of computational models capable of accurately evaluating binding affinity remains one of the main goals of modern computational biophysics. In this work, free energy perturbation/molecular dynamics simulations were used to evaluate absolute and relative binding affinity for three different antidepressants to a sodium-dependent membrane transporter, LeuT, a bacterial homologue of human serotonin and dopamine transporters. Dysfunction of these membrane transporters in mammals has been implicated in multiple diseases of the nervous system, including bipolar disorder and depression. Furthermore, these proteins are key targets for antidepressants including fluoxetine (aka Prozac) and tricyclic antidepressants known to block transport activity. In addition to being clinically relevant, this system, where multiple crystal structures are readily available, represents an ideal testing ground for methods used to study the molecular mechanisms of ligand binding to membrane proteins. We discuss possible pitfalls and different levels of approximation required to evaluate binding affinity, such as the dependence of the computed affinities on the strength of constraints and the sensitivity of the computed affinities to the particular partial charges derived from restrained electrostatic potential fitting of quantum mechanics electrostatic potential. Finally, we compare the effects of different constraint schemes on the absolute and relative binding affinities obtained from free energy simulations.

Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters.

The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.

Transporting Mechanisms of Sodium-Dependent Secondary Membrane Transporters: Insights form Computational Simulations

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding motifs and a well-conserved inverted-repeat topology between proteins from several gene families. Molecular Dynamics and free energy simulations are applied to study the mechanisms of selective binding of ion and substrate in LeuT, vSGLT, and Mhp1. We found that water molecules accessed from periplasm and cytoplasm to the binding sites play an important role in affinity and selectivity of the ion/substrate binding. String method is applied to study the transition from outward-facing state to inward-facing state, providing a minimal-free energy pathway of the transition. These studies identify the sequence of ion and substrate binding/releasing associated with the transporting cycle and provide microscopic mechanical mechanisms of gating.

Mechanism of Selective Cation Binding to Sodium-Coupled Transporters: Insights from Free Energy Simulations and QM/MM Simulations

Ion-coupled transport of neurotransmitter molecules by secondary amino-acids transporters plays pivotal role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling, specificity for a particular cation and the corresponding ion-substrate stoichiometry in secondary transporters has yet to be understood. We have studied Li+/K+/Tl+/Na+ binding and/or selectivity to several transporters with available crystal structures such as the bacterial aspartate transporter GltPh, leucing transporter LeuT and maltose transporter vSGLT using free energy simulations and QM/MM minimization to evaluate the role of different factors in the observed selectivity and ion binding to the protein. Two different mechanisms were found to co-exist for crystallographically characterized binding sites Na1 and Na2 in LeuT and Glt. Furthermore, site Na1 appeared to be well conserved amongst members of different families. To evaluate the role of Na+ binding in the transporter function, we have performed free energy simulations to determine actual cation selectivity as well as binding affinity for sites Na1 and Na2 in the protein. QM/MM minimization was used to characterize the role of the electronic effects of the stabilization of non-native cations such as Li+ and Tl+ in the Na+-selective sites of different transporters. In the case of Tl+ binding to Glt transporter, neighboring residues from a second solvation shell provide a necessary stabilization to the larger cation due to polarization and charge transfer effects implying a rather large flexibility of the metal binding sites.