Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results.</p

Bile Acids Gate Dopamine Transporter Mediated Currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764\u201325773; Cheng and Bahar, Structure, 2015, 23, 2171\u20132181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors

Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results. © 2014 Das et al

Conformational states visited by LeuT during its transport cycle and corresponding hydration patterns and changes in interactions at IC and EC gates.

<p>(<b>A</b>) Six states, labeled, are distinguished, including three newly determined ones: <i>holo-occluded</i>, inward-facing substrate-bound open (IF<i>o</i>*), and <i>apo-occluded</i>. The association/dissociation of the two putative IC gating pairs, R5-D369 and W8-Y268-Y265 (shown in <i>licorice</i>), distinguishes the OF and IF states, along with changes in TM1 and TM6 orientations. Hydrated regions are indicated by <i>blue</i> shaded areas. (<b>B</b>) Two EC gates R30-D404 and F253-Y108 exhibit <i>closed</i> or <i>open</i> (indicated by <i>red dashed line</i>) conformations depending on the LeuT state. At least one of the EC gates is closed in all states, except in OF<i>o</i>. In <i>holo-occluded</i> and <i>apo-occluded</i> states, the substrate binding site is practically occluded to both EC and IC environments, with at least one EC gate and one IC gate being closed concurrently.</p

Theoretical studies of the M2 transmembrane segment of the glycine receptor: Models of the open pore structure and current-voltage characteristics

The pentameric glycine receptor (GlyR), a member of the nicotinicoid superfamily of ligand-gated ion channels, is an inhibitory Cl- channel that is gated by glycine. Using recently published NMR data of the second transmembrane segment (M2) of the human α1 GlyR, structural models of pentameric assemblies embedded in a lipid bilayer were constructed using a combination of experimentally determined constraints coupled with all-atom energy minimization. Based on this structure of the pentameric M2 pore , Brownian dynamics simulations of ion permeation through this putative conducting open state of the channel were carried out. Simulated I-V curves were in good agreement with published experimental current-voltage curves and the anion/cation permeability ratio, suggesting that our open-state model may be representative of the conducting channel of the full-length receptor. These studies also predicted regions of chloride occupancy and suggested residues critical to anion permeation. Calculations of the conductance of the cation-selective mutant A251E channel are also consistent with experimental data. In addition, both rotation and untilting of the pore helices of our model were found to be broadly consistent with closing of the channel, albeit at distinct regions that may reflect alternate gates of the receptor. © 2005 by the Biophysical Society

Involvement of N-terminal residues R5, E6 and W8 in the stabilization of LeuT OF and IF states.

<p>(<b>A</b>) A snapshot from cMD simulation of LeuT in the OF state, illustrating the cation-π interaction R5-Y268, the salt bridge R5-D369, and a hydrogen bond between W8 and Y268 backbones, which completely obstruct access to substrate-binding site from the IC region. (<b>B</b> and <b>C)</b> The same region in the IF state of LeuT. Two alternative N-terminal conformations, superimposed in (<b>B</b>) and further compared in (<b>C</b>) are observed for the IF<i>o</i> state reached at the end of <i>runs 8</i> (<i>white</i>) and <i>11</i> (<i>gray</i>): conformation 1 (<b>C</b>, <i>top</i>) stabilized by the salt bridge R270-D274 (TM7); and conformation 2 (<b>C</b>, <i>bottom</i>) stabilized by the salt-bridges R5-E192 and/or E6-R193 (not shown), and the cation-π interaction and hydrogen bond between W8 and K196. (<b>D</b> and <b>E</b>) Switches between salt-bridges involving R5 and E6 as the structure evolves from OF<i>c</i>* to IF<i>o</i> in the respective <i>runs 8</i> and <i>11</i>. The R5-D369 (<i>red</i>) and E6-R375 (<i>cyan</i>) salt bridges that close the IC vestibule in the OF<i>c</i>* give way to new salt bridges R5-D274 (<i>magenta</i>) and E6-R270 (<i>brown</i>) characteristic of IF<i>o</i> conformer 1 (<b>D</b>), or to R5-E192 (<i>green</i>) of IF<i>o</i> conformer 2 (<b>E</b>). The EC gate R30-D404 remains closed at all times during the transition OF<i>c</i>*→ <i>holo-occluded</i> →IF<i>o*</i> →IF<i>o</i>.</p

Interhelical distances at different states observed in simulations and in the crystal structures.

<p>* TM1a (R11 to A22), TM1b (L25 to A35), TM6a (G242 to L255), TM6b (F259 to Y268), and TM10 EC half (K398 to V412) C^α atoms were used for the calculations of the center of mass. Results for OF<i>o</i>/OF<i>o</i>* were taken from our previous study (ref 4); results for OFc* were averaged based on <i>runs 1</i> to <i>2</i>; for <i>holo-occluded</i> state, equilibrated conformers in <i>runs 6</i> and <i>7</i> were used; for IF<i>o</i>/IF<i>o</i>*, results were averaged based on <i>runs 8</i> to <i>17</i>; for <i>apo-occluded</i> state, equilibrated conformers in <i>runs 18</i> and <i>19 were</i> taken.</p><p>Interhelical distances at different states observed in simulations and in the crystal structures.</p

Outward-facing (OF) and inward-facing (IF) states of LeuT, displayed in explicit lipid and water molecules.

<p>The panels display the MD set ups of (<b>A</b>) LeuT OF<i>c</i>* (PDB: 2A65; <i>orange</i>) and (<b>B)</b> IF<i>o</i> (PDB: 3TT3; <i>cyan</i>) structures embedded into POPC lipid bilayer (<i>green</i>) and solvated by 0.1 M NaCl (not shown) solution. POPC phosphorus atoms are shown in <i>tan</i> spheres, water molecules in <i>red lines</i>. The <i>blue</i> spheres in (<b>A</b>) represent the two Na⁺ ions immobilized in the crystal structure. The bound Leu in the crystal structure is replaced by Ala (<i>purple</i>) in the simulations. Helices labeled in (<b>B</b>), including the broken helices TM1a-b and TM6a-b, exhibit notable reorientations.</p