Neurotransmitter: Study On The Regulatory Mechanism Of Sodium Cotransporter Homolog LeuT On Substrate Transpor

Neurochemical transmission is one of the most important ways of synaptic transmission in the central nervous system.Neurotransmitters bind to the corresponding postsynaptic membrane receptors immediately after release from the presynaptic membrane,leading to depolarization or hyperpolarization of synaptic membranes,and to increase or decrease the cell excitability.Neurotransmitter:Sodium Sympoter（NSS）family members re-uptake neurotransmitters released into synapses such as biogenic amines（dopamine,norepinephrine and serotonin）,amino acids（GABA,glycine and proline）and osmolytes（betaine and creatine）via a synergistic transport mechanism driven by Na⁺electrochemical gradient to terminate neurotransmitter-mediated neuronal signal transmission.The mechanism of NSS transport is generally described by the thermodynamically coupled binding of substrate and ion to a central binding site that is alternately accessible to either side of the membrane.Leucine transporter（Leu T）,a homologue of NSS from Aquifex aeolicus,has become a model protein for studying the structure and mechanism of NSS transporters due to its high homology in sequence,structure and function with other NSS members.The crystal structure elucidates that the transport activity of all Leu T-fold transporters depends on the conformational cycle from open（inward facing or outward facing）to substrate occluded states,as well as structural isomerization events that alternately open and close extracellular and intracellular gates to sequentially open and occlude the central S1 binding site to allow substrate and ions to bind and release across the membrane.Hence,the crystal structures of typical Leu T-fold transporters from multiple superfamilies are defined as inward-facing state,outward-facing state and substrate occluded state,and the conformational changes of the transport cycle are accompanied by the movement of Na⁺along the electrochemical gradient and the uphill movement of substrates.Studies have shown that Na⁺plays a vital role in the conformational cycle.Na⁺increases the affinity of the substrate bound to the central S1 site and promotes the substrate binding in the outward facing conformation.The absence of Na⁺increases the accessibility of intracellular region,promoting the formation of inward-open conformation of Leu T and substrate release.In addition,Na⁺binding,which stabilizes the inward-occluded state and promotes substrate-induced dynamics of the intracellular gate.However,when Li⁺replaces Na⁺,it exhibits a similar stoichiometric ratio（2:1）between the substrate and Leu T,stabilizes the inward-occluded conformation,but does not support substrate Ala-induced intracellular gate dynamics and reduce the activation barrier for the closed-to-open transition by the intracellular gate.To explain this observation,and to further clarify the interaction among substrates,ion binding sites and their adjacent amino acid residues,as well as the regulatory network of substrates binding and transport driven by Na⁺coupling,in this study,Leu T was expressed and purified from E.coli containing an N-terminal His₁₀-tag expression vector,using DDM extraction with Na⁺-free Li⁺buffer,Co²⁺-NTA beads and gel-filtration chromatography.Besides,Leu T displays stable and mono-dispersed peaks when tested in different detergents including DM,NM,LDAO,NG and OG by gel-filtration chromatography.Vapor diffusion method and various commercial crystallization screening kits were used for protein crystallization trials.The diffraction of crystals was improved by optimizing protein concentration,precipitatant concentration and buffer p H.Crystals were cryoprotected by gradually increasing the precipitatant concentration before flash-frozen in liquid N₂ and tested at the Shanghai Synchrotron Radiation Facility.Eventually,high-resolution crystal structures of Leu T/Li⁺/OG with different substrates Ala,Leu,Met and Se Met were solved.These structures assume an outward-facing occluded conformation and show that two Li⁺ions occupy Na1 and Na2 sites,and Li⁺does not cause significant structural changes of Leu T compared to that with Na⁺bound.Based on our structures,site-directed mutagenesis of Leu T substrate binding sites S1 and S2,³H-labeled substrate transport experiments and proximity scintillation analysis were performed.Our results show that no significant effect on the binding of Ala to the S1 and S2 sites was observed after Li⁺replaces Na⁺,but weakened coordination between Li⁺and substrates Ala and Leu was observed,which destroyed the high affinity binding of Leu at the S1 site.In addition,the results of molecular dynamics simulations with polarizable force field and binding free energy calculations illustrated that the existence of a Na1’site near by Na1,and the negatively-charged side chain of Glu290 and the polar side chain of Asn286were mainly involved in the coordination.Li⁺rapidly separates from Na1 site and binds preferentially to Na1’site,leading to rearrangement of substrate in S1 site,and consequently resulting in significant impairment of Leu binding.Also,the favorable site for Li⁺is Na1’,not Na1,which explains why Li⁺doesn’t support Ala transport.Moreover,Li⁺bound at Na2 site results in partial loss of stable coordination between Na2 and Gly20/Val23,which disrupts the position of the carbonyl oxygen of Ala22 and the structural coupling between substrate bound to the S1 site and the ion-binding（Na1 and Na2）sites,suggesting that the physicochemical properties of Li⁺not only weaken the interaction between the cation and substrate in the S1 site,but also severely impact the integrity of the allosteric interaction network that connects the Na⁺and substrate binding sites.Neurotransmitter transporters are important therapeutic targets for many psychiatric diseases and drug addiction.At present,a series of transport inhibitors and substrate analogues have been developed for different NSS family members,including Tricyclic Antidepressants（TCA）,Selective Serotonin Reuptake Inhibitors（SSRIs）,Serotonin/Noradrenaline Reuptake Inhibitors（SNRIs）and selective noradrenaline re-uptake Inhibitors（NRIs）.These inhibitors are used to block the re-uptake of neurotransmitters in synaptic space,thus achieving the goal of treating depression,autism,migraine and other psychiatric diseases.A large number of studies on the kinetics and crystallography of these inhibitors have revealed the existence of competitive and non-competitive inhibition modes.Competitive inhibitors block conformational cycle and inhibit substrate transport by competing with substrate to bind to the central substrate binding site S1.In the structure of tryptophan-Leu T complex,the large side chain indole ring of tryptophan prevents the closure of transmembrane helix TM 1b,TM 6a and TM 2,leading to the formation of the outward-open conformation.In recent years,the structure of d DAT-ligand complexes has further elucidated the inhibition mechanism.All d DAT-ligand structures are in an outward-open conformation except that in the presence of the substrate analog DCP,which is similar to the substrate-bound occluded state in Leu T.When DCP binds to d DAT,the Phe319 residue is flipped toward Tyr124,partially restricts solvent access to the central substrate binding site S1.However,the distance between the side chains of these Tyr and Phe residues is ¹0 （?） in both d DAT and h SERT structures,suggesting that the bulky inhibitors interfere with the closure of this gate and thereby lock transporters in an outward-open conformation.Similarly,Leu T based structural studies reveal the mode of action of non-competitive inhibitors.The structure of Leu T/TCAs complex showed that TCAs bind in an extracellular-facing vestibule about 11 （?） above the substrate and Na+,just extracellular of the conserved Arg30/Asp404 pair.Intriguingly,its presence triggers a flip in the guanidium group of Arg30 so that the latter forms a direct salt bridge with Asp404,stabilizing the extracellular gate in a closed conformation.In addition,many kinetic data further confirmed that binding of TCAs to S2 site greatly reduces the dissociation rate of 3H-Leu from Leu T,and hampers the intracellular gate dynamics induced by substrate Ala.The complex structures of Leu T with SSRIs（Sertraline,R-fluoxetine and S-fluoxetine）and eukaryotic h SERT-S-citalopram also support this notion.However,due to the fact that the crystal structure of any substrate bound to the S2 allosteric binding sites has not yet been obtained,the existence of S2 site has attracted much controversy.The Leu T-S2 acts as an allosteric trigger for the release of S1 substrate and Na+ into the intracellular environment and plays a pivotal role in the whole conformational cycle.Previous studies have shown that binding of substrates or inhibitors to Leu T-S2 site could trigger two distinct results.Contrary to TCAs inhibitors,molecular dynamics simulations and single-molecule fluorescence resonance energy transfer（sm FRET）experiments demonstrate that binding of substrate Ala to Leu T-S2 site facilitates both the opening of the intracellular gate and its subsequent closure by reducing the activation barrier for such conformational transitions,as well as shortens the inward-closed and the inward-open lifetime.To obtain crystal structures of Leu T with substrate bound to S2,we screened a series of nanobodies（Nb489,Nb494,Nb499 and Nb509）which specifically bind Leu T and stabilize its conformation.Surprisingly,3H-Ala uptake assay revealed that Nb494 inhibits Ala transport by Leu T,suggesting that Nb494 may act as a reuptake inhibitor and play a role in improving antidepression treatment.By contrast,Nb489,Nb499 and Nb509 showed significant enhancements in Ala transport,suggesting that Nb489,Nb499 and Nb509 exert an agonist effect during transmitter re-uptake.This novel observation has not been reported previously and is of great significance for treatment of neurological/psychiatric diseases caused by monoaminergic hyperfunction in clinic.To elucidate the structural mechanism of Nb regualtion,we expressed a large number of Leu T and Nb proteins,and screened detergents and conditions suitable for crystallization.Lipid cubic phase and vapor diffusion methods were performed to screen protein crystallization conditions.Various commercial crystallization screening kits were used for crystal screening,and crystals were optimized by optimizing protein concentration,p H,precipitant concentration,and utilizing additive screens.Finally,the structures of Nb489-Leu T and Nb509-Leu T complexe were determined by X-ray diffraction.The structures show that Nb489/Nb509 bind to the extracellular vestibule of Leu T,with complementary decision region CDR3 of Nb489/Nb509 extending into the transport channel of Leu T but not affecting its solvent entrance.Nb489-W106 or Nb509-L106 residue occupies the S2 site,overlapping with TCAs and OG in previously published structures.Furthermore,a series of Nb-Leu T structures were obtained by adding substrates of different sizes,and only S1 site contains substrate.Residue 106 of Nb489/Nb509 bound to Leu T-S2 site exerts the function of allosteric regulation for the release of S1 substrate and Na+.The mechanism of transport potentiation may involve the following aspects:（1）It accelerates the transition of Leu T from the inward-facing to outward-facing state;（2）It promotes the intracellular dynamics leading to faster S1 substrate release;（3）It accelerates S1 binding.