Zhijian Huang

Dynamics of the Extracellular Gate and Ion-Substrate Coupling in the Glutamate Transporter

Glutamate transporters (GluTs) are the primary regulators of extracellular concentration of the neurotransmitter glutamate in the central nervous system. In this study, we have investigated the dynamics and coupling of the substrate and Na(+) binding sites, and the mechanism of cotransport of Na(+) ions, using molecular dynamics simulations of a membrane-embedded model of GluT in its apo (empty form) and various Na(+)- and/or substrate-bound states. The results shed light on the mechanism of the extracellular gate and on the sequence of binding of the substrate and Na(+) ions to GluT during the transport cycle. The results suggest that the helical hairpin HP2 plays the key role of the extracellular gate for the substrate binding site, and that the opening and closure of the gate is controlled by substrate binding. GluT adopts an open conformation in the absence of the substrate exposing the binding sites of the substrate and Na(+) ions to the extracellular solution. Based on the calculated trajectories, we propose that Na1 is the first element to bind GluT, as it is found to be important for the completion of the substrate binding site. The subsequent binding of the substrate, in turn, is shown to result in an almost complete closure of the extracellular gate and the formation of the Na2 binding site. Finally, binding of Na2 locks the extracellular gate and completes the formation of the occluded state of GluT.

Identification of the Third Na+ Site and the Sequence of Extracellular Binding Events in the Glutamate Transporter

The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na(+) ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt(ph). Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Glt(ph) (the apo state, as well as in the presence of Na(+), the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na(+) ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na(+) ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp(312), Thr(92), and Asn(310), along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na(+) ions, reveals a stable structure--showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na(+) ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events.

Transient formation of water-conducting states in membrane transporters

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.

Visualizing Functional Motions of Membrane Transporters with Molecular Dynamics Simulations

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 realistic representation, which is often needed for understanding fundamental molecular properties. The method has proven to be remarkably successful in capturing molecular events and structural transitions highly relevant to the function and/or physicochemical properties of biomolecular systems. Herein, after a brief introduction to the method of MD, we use a number of membrane transport proteins studied in our laboratory as examples to showcase the scope and applicability of the method and its power in characterizing molecular motions of various magnitudes and time scales that are involved in the function of this important class of membrane proteins.

Chapter 10:Molecular Mechanisms of Active Transport Across the Cellular Membrane

Active transport across the cellular membrane constitutes one of the most fundamental processes of life. Taking advantage of various sources of energy in a cell, e.g., ionic and pH gradients, electrical membrane potential, and ATP hydrolysis, specialized molecular machines known as membrane transporters translocate specific molecular species across the cellular membrane, often against their electrochemical gradients. Elucidation of the molecular mechanisms of these complex machines has long been hampered by lack of sufficient structural information, compounded by the complexity of their mechanisms and the lack of the temporal and spatial resolutions required to study in detail their mechanisms experimentally. Recent advances in structural determination of membrane proteins have resulted in solution of a number of high-resolution structures of membrane transporters setting the stage for simulation studies to investigate various aspects of transport at an atomic level. In this chapter, we report the results of a representative collection of our recent simulation studies performed on a number of membrane transporters for which structures became available recently. The studied transporters are structurally diverse, and, more importantly, function using different mechanisms of energy coupling and structural changes involved in the transport cycle. The studied systems reported in this chapter are: 1) the maltose transporter, representing the superfamily of ABC transporters; 2) the glutamate transporter, a member of the secondary membrane transporter family; 3) glycerol phosphate transporter, representing the major facilitator superfamily; 4) ADP/ATP carrier, a mitochondrial carrier; and, 5) the vitamin B12 transporter, representing outer membrane transporters.

Mechanism of Intracellular Gating in the Glutamate Transporter

Glutamate transporters (GlTs) are membrane proteins that regulate and remove synaptically released neurotransmitter glutamate, and maintain normal excitatory synaptic transmission. The recently solved structure of inward-facing GltPh, a GlT homologue revealed an occluded state with the substrate and two Na+ ions (Na1 and Na2) bound.The inward-facing and outward-facing structures of GltPh have put forward a molecular mechanism by which the transporter mediates Na+-coupled substrate uptake. However, the molecular nature of the intracellular gate and the mechanism of gating are still unknown. Furthermore, the mechanism of release of the substrate and co-transported Na+ ions from their intracellular binding sites remains elusive. We have investigated the transporters dynamics and the coupling between substrate and Na+ ions using an extensive set of molecular dynamics simulations of membrane-embedded model of inward-facing GltPh in various bound states. The results suggest that the helical hairpin HP1 plays the key role of the intracellular gate for the substrate-binding site, and that the opening and closure of the gate is controlled by the Na+ ion in the Na1 site. The Na+ ion in the Na2 site was found to be the first to be released from the inward-facing occluded state and can diffuse into the cytoplasmic solution through the attraction of highly conserved residue Ser65 in TM2. Moreover, upon unbinding of the Na+ ion in the Na1 site, the substrate was observed to completely unbind from the binding site and diffuse into the cytoplasmic solution in our equilibrium simulations along the opening of the intracellular gate HP1. Based on the simulations, we propose that the two structurally resolved Na+ ions release into the cytoplasm froe the inward-facing Gltph before the substrate.