The human serotonin transporter (hSERT) is a member of the neurotransmitter:sodium symporter (NSS) family that mediates active reuptake of serotonin from the synapse into the presynaptic neuron. During the transport cycle, hSERT alternates between outward-facing (OF) and inward-facing (IF) states to translocate its substrate between the two sides of the membrane. During the OF-to-IF state transition, serotonin (aka, 5-HT) is inwardly symported together with Na+ and Cl- ions. The return to the OF state is facilitated by cytosolic K+ binding, a step that is also proposed to act as a kinetic decision point by frustrating the outward transport of 5-HT in the direction opposite to the physiological direction of the cycle. However, as opposed to the Na+ ions, the mechanism of K+ binding, its binding site and regulation have not been thoroughly studied. Moreover, recent studies have challenged the conventional transport stoichiometry (1 5-HTin:1 Nain +:1 Clin -:1 Kout +) in hSERT, suggesting that Cl- might remain bound to the transporter during the entire cycle. To explore the role of cytosolic K+ binding to IF hSERT, we performed an extensive set of molecular dynamics simulations. Starting from the post-release IF conformation and in the presence of cytosolic K+, we generated 50 independent trajectories, each for 200 ns to study the behavior of ions. In more than half of the simulations, spontaneous K+ binding was observed at the Na2 site, a conserved cation-binding site in NSS transporters that has been implicated in controlling conformational transitions. Markov state model analysis of coupled ion dynamics quantifies K+ binding kinetics and identifies K+ occupancy of the Na2 site, with Na+ retained at Na1, as the thermodynamically dominant post-release state. In addition, Cl- remains bound to hSERT in the majority of sampled simulations, consistent with recent experimental observations and suggesting a limited role of Cl- release during this stage of the transport cycle. Together, these results provide a kinetic and mechanistic framework for understanding cytosolic K+ binding to hSERT and its potential role in facilitating the IF-to-OF transition that resets the transport cycle.

Serotonin transporter (SERT) regulates serotonergic signals by reuptaking serotonin from the synaptic clefts back into the presynaptic neurons. The recent resolution of the serotonin-SERT complex in multiple conformational states outlined the complete serotonin import cycle. However, a detailed functional appreciation of SERT also involves deciphering the coupling between global structural changes in the transport cycle to the bound chemicals to be transported. By employing molecular dynamics (MD) simulations and free energy calculations in different ligand binding states, here, we reveal how serotonin binding to SERT initiates the global conformational changes essential for serotonin import. Only when serotonin is bound to the central binding site, wedged between transmembrane helices (TMs) 3 and 8, can the system form an interaction network that bridges the two helical domains of the protein, thereby promoting the closure of an extracellular hydrophobic gate and sealing the bound serotonin. To test the role of this hydrophobic gate closure, we designed a series of nonequilibrium MD simulations to steer the outward-facing ↔ occluded transition with different gating configurations. The difference in nonequilibrium work required to fuel the transition indicates that the transition is more likely to happen when the extracellular gate is closed. The transition is not promoted when the gate is open or when 5-HT moves away from TM3 and TM8 toward an alternate pose. Such a local-global coupling is likely shared by other monoamine transporters considering the conservation of all involved structural elements.

A detailed understanding of ligand-protein interaction is essential for developing rational drug-design strategies. In recent years, technological advances in cryo-electron microscopy (cryo-EM) brought a new era to the structural determination of biological macromolecules and assemblies at high resolution, marking cryo-EM as a promising tool for studying ligand-protein interactions. However, even in high-resolution cryo-EM results, the densities for the bound small-molecule ligands are often of lower quality due to their relatively dynamic and flexible nature, frustrating their accurate coordinate assignment. To address the challenge of ligand modeling in cryo-EM maps, here we report the development of GOLEM (Genetic Optimization of Ligands in Experimental Maps), an automated and robust ligand docking method that predicts a ligand's pose and conformation in cryo-EM maps. GOLEM employs a Lamarckian genetic algorithm to perform a hybrid global/local search for exploring the ligand's conformational, orientational, and positional space. As an important feature, GOLEM explicitly considers water molecules and places them at optimal positions and orientations. GOLEM takes into account both molecular energetics and the correlation with the cryo-EM maps in its scoring function to optimally place the ligand. We have validated GOLEM against multiple cryo-EM structures with a wide range of map resolutions and ligand types, returning ligand poses in excellent agreement with the densities. As a VMD plugin, GOLEM is free of charge and accessible to the community. With these features, GOLEM will provide a valuable tool for ligand modeling in cryo-EM efforts toward drug discovery.

The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here, we develop methods to isolate SERT from porcine brain (pSERT) using a mild, nonionic detergent, utilize fluorescence-detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to the central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to the pSERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules.

Degradation behavior of silicon-modified aluminide coating over 30,000 h aging at 650 degrees C was investigated. Significant microstructural evolutions, abnormal elemental distribution and newly-formed secondary internal oxides were observed after prolonged aging. As the aging time extended from 10,000 h to more than 20,000 h, interdiffusion induced the formation of alpha-Fe/AlNi/alpha-Fe structure, while the AlNi precipitates gradually replaced the Fe-rich matrix by acting as Al source for the formation of surface alumina scale. Based on the microstructural evolutions, a new regime of coating degradation involving both the transitions of diffusion mode and oxidation mechanism is proposed.

Structures and membrane interactions of native serotonin transporter in 1 complexes with psychostimulants 2 3 Dongxue Yang1,4, Zhiyu Zhao3, Emad Tajkhorshid3, and Eric Gouaux1,2 4 5 1. Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, 6 USA. 7 2. Howard Hughes Medical Institute, Oregon Health & Science University, Portland, 8 Oregon 97239, USA. 9 3. Department of Biochemistry, NIH Center for Macromolecular Modeling and 10 Bioinformatics, Beckman Institute for Advanced Science and Technology, and Center 11 for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, 12 Urbana, IL, USA. 13 4. Present address: Department of Urology, Institute of Urology (Laboratory of 14 Reconstructive Urology), West China Hospital, Sichuan University, Chengdu, Sichuan, 15 China. 16

Prestin is a high-density motor protein in the outer hair cells (OHCs), whose conformational response to acoustic signals alters the shape of the cell, thereby playing a major role in sound amplification by the cochlea. Despite recent structures, prestin's intimate interactions with the membrane, which are central to its function remained unresolved. Here, employing a large set (collectively, more than 0.5 ms) of coarse-grained molecular dynamics simulations, we demonstrate the impact of prestin's lipid-protein interactions on its organization at densities relevant to the OHCs and its effectiveness in reshaping OHCs. Prestin causes anisotropic membrane deformation, which mediates a preferential membrane organization of prestin where deformation patterns by neighboring copies are aligned constructively. The resulting reduced membrane rigidity is hypothesized to maximize the impact of prestin on OHC reshaping. These results demonstrate a clear case of protein-protein cooperative communication in membrane, purely mediated by interactions with lipids.

Prestin, the electromotive protein found in outer hair cells (OHCs), is responsible for sound amplification without which the hearing system is dysfunctional. Known as an “incomplete transporter”, prestin transits between an expanded and a contracted state in response to membrane potential shifts, resulting in sudden alteration of the surface area in OHCs. Although prestin was discovered decades ago, its molecular mechanism and unique interactions with lipids to control the surface area in OHCs remained elusive until recently. The first atomic-resolution structures of prestin, identified by our collaborators for both the contracted and expanded forms, allowed for molecular exploration. To provide microscopic insight into prestin’s function, we employed all-atom molecular dynamics (MD) simulations of the protein’s states in lipid bilayers. The simulations reveal that prestin substantially twists and curves the membrane, indicating that membrane deformation is a major component of the configurational changes induced by prestin’s conformational dynamics. To directly study the change in the membrane cross-sectional area associated with conformational transition, prestin’s structural transitions were induced using non-equilibrium MD simulations, observing a membrane surface area changes around 3-4%, in close agreement with experimental studies. To further investigate into the collective effect of prestin copies on the surface of OHCs, we employed coarse-grained MD simulations to sample multiple copies of prestin simultaneously in a lipid environment, at varying initial orientations and distances. The systems with all prestins in either conformations exhibit distinct membrane curvature patterns and total membrane surface areas. The membrane bending energy calculation suggests that prestin copies favor specific relative orientations in each conformation, with contracted copies being more perpendicular with respect to each other. Overall, our simulations suggest that prestin conformational changes and prestin-prestin membrane-mediated interactions can contribute to surface area changes in OHCs and hearing signal amplification.

Microstructural evolution of silicon-modified aluminide coating during 10,000 h aging at 650 degrees C was systematically investigated. Upon aging, the coating was gradually transformed from a multilayered structure to a double-layer structure, with the matrixes of Al-Fe-Si compounds and beta-(Fe, Ni)Al in the two layers, respectively. An internal oxide layer formed below the coating, while the thickness of interdiffusion zone (IDZ) increased as the inward diffusion of Al. Precipitate evolutions in both the coating and the IDZ were discussed, which clarifies the microstructural origin of long-term stability of the coating. The phase evolution of Al-Si coating in prolonged aging was further discussed.

The mechanism of fatigue-induced interface damage in Cu/V nanoscale metallic multilayers was systematically investigated using the state-of-the-art in situ transmission electron microscopy. Upon cyclic compression, Cu/V nanoscale metallic multilayers show an interface-controlled fatigue deformation, and the interface morphology of Cu/V nanopillars changes significantly after 200 cyclic loadings. Cyclic deformation induced nanoscale intrusions/extrusions on the bimetal interface, while no sign of persistent slip bands or crack was observed. Formation of the nano-intrusions/extrusions can be attributed to the reciprocating defect motion and severe defect-interface interactions during the fatigue testing. This study provides a unique mechanistic understanding on the fatigue damage in metallic multilayers at nanoscale and sheds light on tailoring the damage tolerance of heterostructures via interface engineering.

Nanoscale materials modified by crystal defects exhibit significantly different behaviours upon chemical reactions such as oxidation, catalysis, lithiation and epitaxial growth. However, unveiling the exact defect-controlled reaction dynamics (e.g. oxidation) at atomic scale remains a challenge for applications. Here, using in situ high-resolution transmission electron microscopy and first-principles calculations, we reveal the dynamics of a general site-selective oxidation behaviour in nanotwinned silver and palladium driven by individual stacking-faults and twin boundaries. The coherent planar defects crossing the surface exhibit the highest oxygen binding energies, leading to preferential nucleation of oxides at these intersections. Planar-fault mediated diffusion of oxygen atoms is shown to catalyse subsequent layer-by-layer inward oxide growth via atomic steps migrating on the oxide-metal interface. These findings provide an atomistic visualization of the complex reaction dynamics controlled by planar defects in metallic nanostructures, which could enable the modification of physiochemical performances in nanomaterials through defect engineering.

Quantum tunnelling offers a unique opportunity to study nanoscale objects with atomic resolution using electrical readout. However, practical implementation is impeded by the lack of simple, stable probes, that are required for successful operation. Existing platforms offer low throughput and operate in a limited range of analyte concentrations, as there is no active control to transport molecules to the sensor. We report on a standalone tunnelling probe based on double-barrelled capillary nanoelectrodes that do not require a conductive substrate to operate unlike other techniques, such as scanning tunnelling microscopy. These probes can be used to efficiently operate in solution environments and detect single molecules, including mononucleotides, oligonucleotides, and proteins. The probes are simple to fabricate, exhibit remarkable stability, and can be combined with dielectrophoretic trapping, enabling active analyte transport to the tunnelling sensor. The latter allows for up to 5-orders of magnitude increase in event detection rates and sub-femtomolar sensitivity.

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC ( Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C—at the agonist-binding sites—and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD–TMD coupling in such a way that the “conformational wave” of liganded-receptor gating takes place in the ECD and the interfacial M2–M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand’s fluctuations, and thus delays its escape into bulk solvent.

The human serotonin transporter (hSERT) belongs to the neurotransmitter:sodium symporter (NSS) family, which regulate neurotransmission by re-uptaking the released neurotransmitters into the presynaptic neuron. During the transport cycle, the transporter alternates between the outward-facing (OF) and inward-facing (IF) states to translocate the substrate across the membrane. Central to the cycle in hSERT is the OF-to-IF state transition, during which 5-HT (serotonin) is inwardly symported in a Na+- and Cl--dependent manner, followed by cytosolic-K+-binding-triggered returning to the OF state. K+ binding might act as a "kinetic decision point" by frustrating the outward transport of 5-HT, namely, the reverse of the physiological direction. However, the mechanism of such K+ regulation remains elusive. Moreover, the conventional transport stoichiometry (15-HTin:1Na+in:1Cl−in:1K+out) has been challenged by recent studies showing that Cl- might remain bound to hSERT during the entire cycle. To explore the role of cytosolic K+ binding to IF hSERT, we have constructed a Markov State Model from an extensive set of molecular dynamics simulations. Starting with the IF state, the system is modeled under turnover conditions, i.e., post-5-HT-release and exposed to cytosolic K+ concentration, resulting in 50 independent, 200-ns replicas. More than half of the trajectories capture spontaneous K+ binding to the Na2 site, a conserved Na+ binding site within NSSs deemed to control the transporter conformational changes and substrate release. Furthermore, consistent with recent experimental results, Cl- was found to remain bound in the majority of the ensembles, questioning the contribution of the Cl- gradient to the substrate transport. Together, these results characterize the previously unknown K+ site in hSERT and present a quantitative kinetic description of K+ binding, shedding light on the mechanism of the ion-dependence of the IF-to-OF transition that resets the transport cycle.

Membrane transporters are key gatekeeper proteins at cellular membranes that closely control the traffic of materials. Their function relies on structural rearrangements of varying degrees that facilitate substrate translocation across the membrane. Characterizing these functionally important molecular events at a microscopic level is key to our understanding of membrane transport, yet challenging to achieve experimentally. Recent advances in simulation technology and computing power have rendered molecular dynamics (MD) simulation a powerful biophysical tool to investigate a wide range of dynamical events spanning multiple spatial and temporal scales. Here, we review recent studies of diverse membrane transporters using computational methods, with an emphasis on highlighting the technical challenges, key lessons learned, and new opportunities to illuminate transporter structure and function.

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons(1-3). Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action(2,4). The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines(2,3), and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder(2,5). Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane(1,6-12). However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties(13,14.) Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors(15,16). Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

Secondary active transporters use electrochemical gradient of ions to fuel the "uphill" translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF ↔ IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF ↔ IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.

Membrane fusion is mediated by SNARE proteins, and is vital for a multitude of cellular transport phenomena. Post fusion, cis-SNAREs are recycled for continued fusion, in an ATP dependent process called priming. The unique job of priming all cellular SNAREs is the responsibility of AAA+ ATPase NSF or Sec18. In a joint computational and experimental effort, we show that Phosphatidic Acid (PA) inhibits the priming of cis-SNARE complexes by inducing conformational changes in Sec18 protomer. We further studied priming inhibition via a new small molecule inhibitor of Sec18 called IPA, which inhibits fusion, priming, and competitively inhibits Sec18 binding to PA. We identify potential PA binding sites to Sec18 using computational flooding of short tailed PA, as well as using an HMMM PA rich membrane to capture unbiased spontaneous membrane insertion of Sec18 monomers. In addition, to further sample potential binding sites of both PA as well as IPA on Sec18 we employed ensemble molecular docking using AutoDock Vina, with consecutive molecular dynamics simulations performed for the top poses using NAMD for characterization of ligand stability. Finally, we performed Random Accelerated Molecular dynamics simulations on the most stable resultant poses to estimate dissociation constants, which were compared to results from liposome binding experiments providing insight into the mechanism of priming regulation via Sec18.