Constraints imposed by the membrane selectively guide the alternating access dynamics of the glutamate transporter GltPh - PubMed (original) (raw)

Constraints imposed by the membrane selectively guide the alternating access dynamics of the glutamate transporter GltPh

Timothy R Lezon et al. Biophys J. 2012.

Abstract

Substrate transport in sodium-coupled amino acid symporters involves a large-scale conformational change that shifts the access to the substrate-binding site from one side of the membrane to the other. The structural change is particularly substantial and entails a unique piston-like quaternary rearrangement in glutamate transporters, as evidenced by the difference between the outward-facing and inward-facing structures resolved for the archaeal aspartate transporter Glt(Ph). These structural changes occur over time and length scales that extend beyond the reach of current fully atomic models, but are regularly explored with the use of elastic network models (ENMs). Despite their success with other membrane proteins, ENM-based approaches for exploring the collective dynamics of Glt(Ph) have fallen short of providing a plausible mechanism. This deficiency is attributed here to the anisotropic constraints imposed by the membrane, which are not incorporated into conventional ENMs. Here we employ two novel (to our knowledge) ENMs to demonstrate that one can largely capture the experimentally observed structural change using only the few lowest-energy modes of motion that are intrinsically accessible to the transporter, provided that the surrounding lipid molecules are incorporated into the ENM. The presence of the membrane reduces the overall energy of the transition compared with conventional models, showing that the membrane not only guides the selected mechanism but also acts as a facilitator. Finally, we show that the dynamics of Glt(Ph) is biased toward transitions of individual subunits of the trimer rather than cooperative transitions of all three subunits simultaneously, suggesting a mechanism of transport that exploits the intrinsic dynamics of individual subunits. Our software is available online at http://www.membranm.csb.pitt.edu.

Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

OF and IF structures of GltPh show a large displacement of the core domains. Each subunit of the homotrimer contains a trimerization domain (wheat) and a transport domain (blue and pink). (A) In the OF conformation (PDB code:

2NWL

), the transport domains extend into the EC medium, forming an aqueous basin around the trimer interface. (B) In the IF conformation (PDB code:

3KBC

), the transport domains are displaced across the membrane to the CP, whereas the trimer interface experiences little conformational change. The top diagrams in panels A and B represent the views from the EC region, and the lower diagrams display the side views within the membrane. Lipid molecules are represented by simple polar heads and hydrophobic tails. Molecular representations were rendered in VMD (47).

Figure 2

Figure 2

Many of the details of the GltPh transport cycle have been resolved. This cartoon summarizes the process for a single subunit. Starting from the OF unbound state (A), the flexible HP2 loop opens (B), exposing the sodium and substrate-binding sites. Three Na+ ions and the substrate bind (C), starting from the binding of a first Na+ (to the Na3 site), and then bind to a substrate and a sodium ion (to the Na2 site), and finally to the Na1 site. Here, for simplicity, we do not show these intermediate steps. The HP2 loop closes (D) and the transport domain moves through the membrane to the cytoplasm, placing the subunit in the IF closed state (E). The HP2 loop again opens (F), permitting the release of one Na+ ion from the Na1 site and subsequent hydration of the binding pocket. Water molecules aid in substrate dissociation by promoting the opening of HP1 and flooding the binding site, eventually leading to exit of substrate and the remaining Na+ ions (G). When the HP loops close (H), the transport domain moves back across the membrane to the EC face, completing the cycle. The global conformational changes D-E and G-A are the focus of this study.

Figure 3

Figure 3

First three modes of _im_ANM suggest a mechanism for conformational change. (A) The slowest nondegenerate ANM mode for the OF conformation of GltPh (top) is a pincer-type opening/closing of the trimer through motions of the transport domains. The same mode in the IF conformation (bottom) is a coordinated twisting of the transport domains. (B) In _im_ANM, the first nondegenerate mode is a piston-type axial motion of the transport domains for both the OF and IF conformations. It allows both the OF and IF conformations to move toward a transition intermediate, indicated by the arrows at far right. In all cases, the first twofold degenerate mode is the same as the mode shown, but with one of the subunits phase-shifted by 180°. (C) The transition for a single subunit. Starting from the OF crystal structure at far left, the motion along the first nondegenerate mode brings the transport domain into the membrane, as shown by the blue arrow. Starting from the IF crystal structure at right, the slowest nondegenerate mode moves the transport domain up toward the EC region. The change that is not captured by this mode is the difference between the two centermost structures. HP1 and HP2 are colored yellow and red, respectively.

Figure 4

Figure 4

Separation of each subunit into transport and trimerization domains is visualized by using the cosines of the angles between residue motions (Eq. S4). Each matrix element indicates the cosine of the angle between motions of two residues, as calculated from the second mode of _im_ANM (A and B) or ANM (C and D). Red indicates comoving (fully correlated) residues, and blue indicates oppositely moving (fully anticorrelated) residues. (A) The second _im_ANM mode moves TM1, TM2, TM4, and TM5 in one direction, and TM3, TM6, TM7, HP2, and TM8 in the opposite direction. The coupling of TM3 and TM6 to the transport domain is contrary to the original domain definition provided by Boudker and co-workers (6). For reference, schematics of the locations of the eight TM helices are also shown, with the transport domain in blue, the trimerization domain in wheat, and the HP loops in dark red. (B) A similar sharp domain separation is seen for _im_ANM mode 2 of the IF conformation. (C and D) The ANM results produce a less clear demarcation.

Figure 5

Figure 5

Inclusion of the membrane improves the efficiency of the modes in reproducing the structural deformation. In the left panels, the cumulative overlap of the deformation vector is plotted against the slowest 50 modes using different ENMs. The ANM (black) shows a steady increase in overlap with the number of modes considered. In contrast, the first nondegenerate mode of the _im_ANM (magenta) accounts for more than half of the deformation in either direction. The _ex_ANM (blue) shows behavior similar to that of the _im_ANM for the OF-IF transition, and ANM-like behavior for the IF-OF transition. A larger overlap can be obtained by increasing the _im_ANM membrane scaling factor, but this results in loss of overlap in higher modes (green). In the right panels, the cumulative overlap is plotted against the normalized energy of deformation between the two conformations. The membrane-augmented models (magenta and blue) allow for greater displacements at lower energies compared with ANM (black). The energetic cost of using the _im_ANM eventually overtakes that of ANM.

Figure 6

Figure 6

Cumulative overlap curves suggest possible transitions to intermediate conformations. Each plot shows the overlap of the first 20 modes of ANM (black), _im_ANM (magenta), and _ex_ANM (blue), with the deformation between the starting structure shown at left and the ending structure shown at top.

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