Atomistic modeling of alternating access of a mitochondrial ADP/ATP membrane transporter with molecular simulations

<div><p>The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.</p></div

Alternating access mechanism of AAC.

<p>(<i>a</i>) X-ray structures in the OF form in views from the matrix side (left) and the cytoplasmic one (right) are shown in upper panels, and the last snapshots of APO1 simulation in the IF form are shown in lower panels. Odd-numbered helices are colored in wheat and even-numbered ones in green, respectively. Residues comprising the cytoplasmic salt-bridge network in the IF form are drawn in stick representation in red for acidic residues and in blue for basic ones. Asp203 and Arg104 are colored in magenta and cyan, respectively. Residues constituting the cytoplasmic hydrophobic packing in the IF form are drawn in gray stick and vdW sphere representations. (<i>b</i>) Hydrophobic residues comprising the matrix hydrophobic core in the OF form and the cytoplasmic one in the IF form are shown in black surface representation. Basic residues participating in the binding of ADP or BA are shown in blue surface representation.</p

Linear Response Path Following: A Molecular Dynamics Method To Simulate Global Conformational Changes of Protein upon Ligand Binding

Molecular functions of proteins are often fulfilled by global conformational changes that couple with local events such as the binding of ligand molecules. High molecular complexity of proteins has, however, been an obstacle to obtain an atomistic view of the global conformational transitions, imposing a limitation on the mechanistic understanding of the functional processes. In this study, we developed a new method of molecular dynamics (MD) simulation called the linear response path following (LRPF) to simulate a protein’s global conformational changes upon ligand binding. The method introduces a biasing force based on a linear response theory, which determines a local reaction coordinate in the configuration space that represents linear coupling between local events of ligand binding and global conformational changes and thus provides one with fully atomistic models undergoing large conformational changes without knowledge of a target structure. The overall transition process involving nonlinear conformational changes is simulated through iterative cycles consisting of a biased MD simulation with an updated linear response force and a following unbiased MD simulation for relaxation. We applied the method to the simulation of global conformational changes of the yeast calmodulin N-terminal domain and successfully searched out the end conformation. The atomistically detailed trajectories revealed a sequence of molecular events that properly lead to the global conformational changes and identified key steps of local–global coupling that induce the conformational transitions. The LRPF method provides one with a powerful means to model conformational changes of proteins such as motors and transporters where local–global coupling plays a pivotal role in their functional processes

Crucial Role of Protein Flexibility in Formation of a Stable Reaction Transition State in an α-Amylase Catalysis

Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic α-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization

Photoactivation Intermediates of a G‑Protein Coupled Receptor Rhodopsin Investigated by a Hybrid Molecular Simulation

Rhodopsin is a G-protein coupled receptor functioning as a photoreceptor for vision through photoactivation of a covalently bound ligand of a retinal protonated Schiff base chromophore. Despite the availability of structural information on the inactivated and activated forms of the receptor, the transition processes initiated by the photoabsorption have not been well understood. Here we theoretically examined the photoactivation processes by means of molecular dynamics (MD) simulations and <i>ab initio</i> quantum mechanical/molecular mechanical (QM/MM) free energy geometry optimizations which enabled accurate geometry determination of the ligand molecule in ample statistical conformational samples of the protein. Structures of the intermediate states of the activation process, blue-shifted intermediate and Lumi, as well as the dark state first generated by MD simulations and then refined by the QM/MM free energy geometry optimizations were characterized by large displacement of the β-ionone ring of retinal along with change in the hydrogen bond of the protonated Schiff base. The <i>ab initio</i> calculations of vibrational and electronic spectroscopic properties of those states well reproduced the experimental observations and successfully identified the molecular origins underlying the spectroscopic features. The structural evolution in the formation of the intermediates provides a molecular insight into the efficient activation processes of the receptor

Opening of the matrix side observed in LRPF2 simulation.

<p>(<i>a-d</i>) Time courses of helix angles (<i>a</i>), distance between Glu29 and Arg279 (<i>b</i>), contact between Phe88 and Tyr290 (<i>c</i>), and contact between Glu29 and the amino group at the adenine ring of ADP (<i>d</i>). See Supporting Material for the definition of the helix angle and the contact. Shaded areas indicate that a time region where a hydrogen-bond between Glu29 and the amino group of the adenine ring was established. (<i>e</i>) Snapshots of LRPF2 simulation at 1900 (left) ns and 2100 ns (right) before and after formation of the hydrogen-bond between Glu29 and the adenine ring, respectively. Close up views of the ADP binding site are shown in right panels.</p

QM/MM Reweighting Free Energy SCF for Geometry Optimization on Extensive Free Energy Surface of Enzymatic Reaction

We developed a quantum mechanical/molecular mechanical (QM/MM) free energy geometry optimization method by which the geometry of a quantum chemically treated (QM) molecule is optimized on a free energy surface defined with thermal distribution of the surrounding molecular environment obtained by molecular dynamics simulation with a molecular mechanics (MM) force field. The method called QM/MM reweighting free energy self-consistent field combines a mean field theory of QM/MM free energy geometry optimization developed by Yamamoto (Yamamoto, T. <i>J. Chem. Phys.</i> <b>2008</b>, <i>129</i>, 244104) with a reweighting scheme for updating the MM distribution introduced by Hu et al. (Hu, H., et al. <i>J. Chem. Phys.</i> <b>2008</b>, <i>128</i>, 034105) and features high computational efficiency suitable for exploring the reaction free energy surface of extensive protein conformational space. The computational efficiency with improved treatment of a long-range electrostatic (ES) interaction using the Ewald summation technique permits one to take into account global conformational relaxation of an entire protein of an enzyme in the free energy geometry optimization of its reaction center. We applied the method to an enzymatic reaction of a substrate complex of psychrophilic α-amylase from Antarctic bacterium Pseudoalteromonas haloplanktis and succeeded in geometry optimizations of the reactant and the product of the catalytic reaction that involve large conformational changes of protein loops adjacent to the reaction center on time scales reaching sub-microseconds. We found that the adjacent loops in the reactant and the product form in different conformations and produce catalytic ES potentials on the reaction center

Typical biasing forces used in LRPF simulations.

<p>(<i>a-b</i>) Typical biasing forces, (<i>a</i>) and (<i>b</i>), applied to C_α-atoms in LRPF2 and LRPF2+ simulations, respectively, are represented by black cones. Protein backbone is drawn in ribbon representation. C_α-atoms to which the perturbative forces, and , are applied are indicated by spheres. The biasing forces were frequently updated during the LRPF simulations.</p

Protein structure of the ADP-bound IF form.

<p>Snapshots at 3,990 ns in MD1 simulation are shown. Hydrophobic residues and charged ones participating in the cytoplasmic inter-domain hydrophobic packing and salt-bridge network in the IF form are shown in views from the cytoplasmic side in left and middle panels, respectively. A side view of the protein structure and the cytoplasmic salt-bridge network is shown in a right panel.</p

Structure of AAC in the OF form and a binding pose of ADP.

<p>(<i>a</i>) An X-ray structure of AAC in the OF form (PDB entry 1OKC). The numbering of bovine AAC isoform 1 starts after the initiating methionine (Ser1–), following the numbering in the PDB file. (<i>b</i>) Schematic illustration of the protein structure. (<i>c</i>) Water distribution in the cytoplasmic pore at the last snapshot of the ADP binding simulation drawn in surface representation and colored in cyan. (<i>d</i>) A binding pose of ADP. Residues 190–230 and 280–297 are not shown. (<i>e</i>) Charged residues participating in ADP binding from the cytoplasmic side. A view from the cytoplasmic side. ADP is not shown.</p