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Title:Molecular insights into alternating access mechanism of secondary active transporters from molecular dynamics simulations
Author(s):Li, Jing
Director of Research:Tajkhorshid, Emad
Doctoral Committee Chair(s):Tajkhorshid, Emad
Doctoral Committee Member(s):Schulten, Klaus J.; Leckband, Deborah E.; Grosman, Claudio F.
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Secondary active transporter
Alternating access mechanism
Molecular dynamics simulations
Ion-coupling mechanism
Water-conducting state
Ion release
Substrate release
LeuT-fold transporters
Abstract:Membrane transporters are specialized molecular machinery for selective, regulable, efficient, and more importantly, active transport of diverse chemical species, e.g., nutrients, reaction precursors and products, and chemicals transmitting signals, across the cellular membrane. As a major class of membrane transporters, secondary active transporters couple vectorial translocation of one solute, typically Na+ or H+ ions, along its electrochemical gradient to uphill transport of their substrates. To fulfill its function, transporter operates via an alternating-access mechanism, in which it undergoes structural transitions between outward-facing (OF) and inward-facing (IF) states, to translocate substrate from one to the other side of the membrane. The alternating access mechanism has received substantial supports from recent structural studies. However, as an inherently dynamic process, several critical aspects of alternating access mechanism remain poorly understood solely on the basis of limited static snapshots of the transport cycle. To address several key questions of alternating access model and to elucidate the underlying molecular mechanism at atomic level, we conducted computational studies mainly using molecular dynamics (MD) simulations on two secondary active transporters, i.e., bacterial Na+–coupled glucose transporter (vSGLT), and the benzyl-hydantoin transporter (Mhp1). vSGLT presents the first IF structural state of a LeuT-fold transporter, and Mhp1 is the first secondary active transporter structurally resolved in both OF and IF states. Both of vSGLT and Mhp1 provided crucial information toward better characterization of the alternating–access mechanism. Based on the crystal structures of these two transporters, the computational studies elucidated several key functional molecular events in the transport cycle, and provided deeper insights of the underlying molecular mechanism of alternating access model. My early study on vSGLT identified the first ion-releasing state in the secondary active transporters. The crystal structure of vSGLT reports the transporter in its substrate-bound state, with a Na+ ion modeled in a binding site corresponding to that of a homologous protein, leucine transporter (LeuT). In repeated MD simulations, however, the Na+ ion is found instable, invariably and spontaneously diffusing out of the transporter through a pathway lined by D189, which appears to facilitate the diffusion of the ion toward the cytoplasm. Further analysis of the trajectories and close structural examination, in particular comparison of the Na+-binding sites of vSGLT and LeuT, strongly indicates that the crystal structure of vSGLT actually represents an ion-releasing state of the transporter. Structural comparison of LeuT-fold transporters provides the first example in which we clearly see how global structural changes (tilting and shift of the helices) that take place during the transition between the IF and OF states, propagate into specific binding site of the ion (expansion of the site), thus, allowing the protein to release the ion into the solution. The observed dynamics of the Na+ ion, in contrast to the substrate, also suggests that the cytoplasmic release of the Na+ ion precedes that of the substrate, thus, shedding light on a key step in the transport cycle of this secondary transporter. The second study on vSGLT is to characterize the next step after ion releasing, the substrate releases to cytoplasm. Employing both equilibrium and Steered MD (SMD) simulations, the pathway and mechanism of substrate unbinding from the IF state of the vSGLT have been investigated. During a 200–ns equilibrium simulation, repeated spontaneous unbinding events of the substrate from its binding site have been observed. In contrast to the previously proposed gating role of a tyrosine residue (Y263), the unbinding mechanism captured in our equilibrium simulation does not rely on the displacement and/or rotation of this side chain. Rather, the unbinding involves an initial lateral displacement of the substrate out of the binding site which allows the substrate to completely emerge from the region covered by the side chain of Y263 without any noticeable conformational changes of the latter. Starting with the snapshots taken from this equilibrium simulation with the substrate outside the binding site, SMD simulations were then used to probe the translocation of the substrate along the remaining of the release pathway within the protein’s lumen and to characterize the nature of protein-substrate interactions involved in the process. Combining the results of the equilibrium and SMD simulations, a full translocation pathway is provided for the substrate release from the binding site into the cytoplasm. The observed molecular events indicate that no gating is required for the release of the substrate from the crystallographically captured IF structure of SGLT, suggesting that this conformation represents an open, rather than occluded, state of the transporter. Although the alternating access 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. and my colleagues performed a large set of extended equilibrium molecular dynamics simulations on several classes of membrane transporters, e.g., vSGLT, Mhp1 and etc., 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 offers an expanded understanding of alternating access mechanism. At the heart of the transport mechanism of these secondary active transporters is the coupling among species providing driving forces, substrate, and the conformational events during the whole transport cycle. To elucidate this crucial and long-term question, a serial of MD simulations were employed to study the impact of Na+–binding on the structure and dynamics of Mhp1 in multiple functional states and on the transition between them. The results of microsecond-long equilibrium MD simulations suggest that Na+ binding stabilizes conformation favorable for the substrate binding in the OF state. Furthermore, the results of a special-protocol time-dependent biased simulation and subsequent free energy calculation for state transition, illustrate that Na+ binding increases the free energy barrier along the OF–IF transition. All the results suggest that Na+ binding reshapes the free-energy landscape of the ion/protein complex, thereby shifting the conformational preference toward a specific OF structure, which is favorable for substrate binding. The increased substrate affinity provided by Na+ binding facilitates uptaking of the substrate from its low-concentration environment by the transporter. The results, therefore, provide a deeper and more comprehensive understanding for the ion-coupling mechanism of secondary active transporters.
Issue Date:2014-05-30
Rights Information:Copyright 2014 Jing Li
Date Available in IDEALS:2014-05-30
Date Deposited:2014-05

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