Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore

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

Structural changes during the formation of early intermediates in the bacteriorhodopsin photocycle.

Early intermediates of bacteriorhodopsin's photocycle were modeled by means of ab initio quantum mechanical/molecular mechanical and molecular dynamics simulations. The photoisomerization of the retinal chromophore and the formation of photoproducts corresponding to the early intermediates were simulated by molecular dynamics simulations. By means of the quantum mechanical/molecular mechanical method, the resulting structures were refined and the respective excitation energies were calculated. Two sequential intermediates were found with absorption maxima that exhibit red shifts from the resting state. The intermediates were therefore assigned to the K and KL states. In K, the conformation of the retinal chromophore is strongly deformed, and the N--H bond of the Schiff base points almost perpendicular to the membrane normal toward Asp-212. The strongly deformed conformation of the chromophore and weakened interaction of the Schiff base with the surrounding polar groups are the means by which the absorbed energy is stored. During the K-to-KL transition, the chromophore undergoes further conformational changes that result in the formation of a hydrogen bond between the N--H group of the Schiff base and Thr-89 as well as other rearrangements of the hydrogen-bond network in the vicinity of the Schiff base, which are suggested to play a key role in the proton transfer process in the later phase of the photocycle

On the Mechanism of ATP Hydrolysis in F(1)-ATPase

Most of the cellular ATP in living organisms is synthesized by the enzyme F(1)F(o)-ATP synthase. The water soluble F(1) part of the enzyme can also work in reverse and utilize the chemical energy released during ATP hydrolysis to generate mechanical motion. Despite the availability of a large amount of biochemical data and several x-ray crystallographic structures of F(1), there still remains a considerable lack of understanding as to how this protein efficiently converts the chemical energy released during the reaction ATP + H(2)O → ADP + P(i) into mechanical motion of the stalk. We report here an ab initio QM/MM study of ATP hydrolysis in the β(TP) catalytic site of F(1). Our simulations provide an atomic level description of the reaction path, its energetics, and the interaction of the nucleotide with the protein environment during catalysis. The simulations suggest that the reaction path with the lowest potential energy barrier proceeds via nucleophilic attack on the γ-phosphate involving two water molecules. Furthermore, the ATP hydrolysis reaction in β(TP) is found to be endothermic, demonstrating that the catalytic site is able to support the synthesis of ATP and does not promote ATP hydrolysis in the particular conformation studied

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

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