Molecular Dynamics Simulation for All.

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

Anton 2 is a second-generation special-purpose supercomputer for molecular dynamics simulations that achieves significant gains in performance, programmability, and capacity compared to its predecessor, Anton 1. The architecture of Anton 2 is tailored for fine-grained event-driven operation, which improves performance by increasing the overlap of computation with communication, and also allows a wider range of algorithms to run efficiently, enabling many new software-based optimizations. A 512-node Anton 2 machine, currently in operation, is up to ten times faster than Anton 1 with the same number of nodes, greatly expanding the reach of all-atom bio molecular simulations. Anton 2 is the first platform to achieve simulation rates of multiple microseconds of physical time per day for systems with millions of atoms. Demonstrating strong scaling, the machine simulates a standard 23,558-atom benchmark system at a rate of 85 µs/day -- 180 times faster than any commodity hardware platform or general-purpose supercomputer.

Anton 2: raising the bar for performance and programmability in a special-purpose molecular dynamics supercomputer

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.

/pdf/x-ray-structures-of-leut-in-substrate-free-outward-open-and-4cey3w6190.pdf

X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.

Acid-sensing ion channels are proton-activated, sodium-selective channels composed of three subunits, and are members of the superfamily of epithelial sodium channels, mechanosensitive and FMRF-amide peptide-gated ion channels. These ubiquitous eukaryotic ion channels have essential roles in biological activities as diverse as sodium homeostasis, taste and pain. Despite their crucial roles in biology and their unusual trimeric subunit stoichiometry, there is little knowledge of the structural and chemical principles underlying their ion channel architecture and ion-binding sites. Here we present the structure of a functional acid-sensing ion channel in a desensitized state at 3 A resolution, the location and composition of the ∼8 A ‘thick’ desensitization gate, and the trigonal antiprism coordination of caesium ions bound in the extracellular vestibule. Comparison of the acid-sensing ion channel structure with the ATP-gated P2X4 receptor reveals similarity in pore architecture and aqueous vestibules, suggesting that there are unanticipated yet common structural and mechanistic principles. P2X receptors are ATP-gated non-selective cation channels involved in nociception and inflammatory responses, whose structures were unknown. Kawate et al. now present the crystal structure of the zebrafish P2X4 receptor in a closed state. The trimeric structure reveals some of the molecular underpinnings of ligand-binding, cation entry and channel gating. A related paper presents the structure of chicken acid-sensing ion channel 1 (ASIC1) in a desensitized state. Like P2X receptors, ASICs are trimeric, but they belong to an entirely different family of ion channels. The structure determination of ASIC1 shows how ion permeation and desensitization may occur, and comparison of ASIC and P2X structures suggests that these functionally distinct channels employ similar mechanistic principles. Like P2X receptors, acid-sensing ion channels are trimeric in structure; however, they belong to an entirely different family. Here, the structure of an acid-sensing ion channel is presented and compared to the structure of P2X4, suggesting that these functionally distinct channels use similar mechanistic principles.

/pdf/pore-architecture-and-ion-sites-in-acid-sensing-ion-channels-4us20mquxn.pdf

Pore architecture and ion sites in acid-sensing ion channels and P2X receptors

Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane—a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mechanical coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equilibrium molecular dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.

https://www.pnas.org/content/pnas/110/19/7696.full.pdf

Transient formation of water-conducting states in membrane transporters

The leucine transporter (LeuT) has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS), and, it exhibits a hallmark 5-TM inverted repeat (“LeuT-fold”), a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of “LeuT-fold” transporters requires detailed structural and dynamic descriptions of the outward-facing (OF) and inward-facing (IF) states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a “LeuT-fold” transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- complex in the IF state, mainly due to TM1 bending. Inducing a transition in only one of the two pseudosymmetric domains, while allowing the second to respond dynamically, is found to be sufficient to induce the formation of the IF state. We also propose that TM2 and TM7 may be facilitators of TM1 and TM6 motion. Thus, this study not only presents a novel modeling methodology applied to obtain the IF state of LeuT, but also describes structural elements involved in a possibly general transport mechanism in transporters adopting the “LeuT-fold”.

/pdf/modeling-and-dynamics-of-the-inward-facing-state-of-a-na-cl-43msw7co17.pdf

Modeling and dynamics of the inward-facing state of a Na+/Cl- dependent neurotransmitter transporter homologue.

Computational modeling and molecular simulation techniques have become an integral part of modern molecular research. Various areas of molecular sciences continue to benefit from, indeed rely on, the unparalleled spatial and temporal resolutions offered by these technologies, to provide a more complete picture of the molecular problems at hand. Because of the continuous development of more efficient algorithms harvesting ever-expanding computational resources, and the emergence of more advanced and novel theories and methodologies, the scope of computational studies has expanded significantly over the past decade, now including much larger molecular systems and far more complex molecular phenomena. Among the various computer modeling techniques, the application of molecular dynamics (MD) simulation and related techniques has particularly drawn attention in biomolecular research, because of the ability of the method to describe the dynamical nature of the molecular systems and thereby to provide a more rea...

https://pubs.acs.org/doi/pdf/10.1021/bi301086x

Visualizing functional motions of membrane transporters with molecular dynamics simulations.

Transmembrane exchange of materials is a fundamental process in biology. Molecular dynamics provides a powerful method to investigate in great detail various aspects of the phenomenon, particularly the permeation of small uncharged molecules, which continues to pose a challenge to experimental studies. We will discuss some of the recent simulation studies investigating the role of lipid-mediated and protein-mediated mechanisms in permeation of water and gas molecules across the membrane.

Exploring transmembrane diffusion pathways with molecular dynamics.

https://www.cell.com/biophysj/pdf/S0006-3495(08)78941-0.pdf

Saher A. Shaikh

Papers

Transient formation of water-conducting states in membrane transporters

Modeling and dynamics of the inward-facing state of a Na+/Cl- dependent neurotransmitter transporter homologue.

Visualizing functional motions of membrane transporters with molecular dynamics simulations.

Exploring transmembrane diffusion pathways with molecular dynamics.

Potential Cation and H+ Binding Sites in Acid Sensing Ion Channel-1