Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsß1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gßγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

Structure of MotA, a flagellar stator protein, from hyperthermophile.

Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotA5MotB2 stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA.

Dissociation Pathways of the p53 DNA Binding Domain from DNA and Critical Roles of Key Residues Elucidated by dPaCS-MD/MSM.

p53 is a transcriptional factor that regulates cell response to a variety of stresses. About a half of all human tumors contain p53 mutations, and the accumulation of mutations in the DNA binding domain of p53 (p53-DBD) can cause destabilization of p53 and its complex with DNA. To identify the key residues of the p53-DBD/DNA binding and to understand the dissociation mechanisms of the p53-DBD/DNA complex, the dissociation process of p53-DBD from a DNA duplex that contains the consensus sequence (the specific target of p53-DBD) was investigated by a combination of dissociation parallel cascade selection molecular dynamics (dPaCS-MD) and the Markov state model (MSM). This combination (dPaCS-MD/MSM) enabled us to simulate dissociation of the two large molecules based on an all-atom model with a short simulation time (11.2 ± 2.2 ns per trial) and to analyze dissociation pathways, free energy landscape (FEL), and binding free energy. Among 75 trials of dPaCS-MD, p53-DBD dissociated first from the major groove and then detached from the minor groove in 93% of the cases, while 7% of the cases unbinding from the minor groove occurred first. Minor groove binding is mainly stabilized by R248, identified as the most important residue that tightly binds deep inside the minor groove. The standard binding free energy calculated from the FEL was -10.9 ± 0.4 kcal/mol, which agrees with an experimental value of -11.1 kcal/mol. These results indicate that the dPaCS-MD/MSM combination can be a powerful tool to investigate dissociation mechanisms of two large molecules. Analysis of the p53 key residues for DNA binding indicates high correlations with cancer-related mutations, confirming that impairment of the interactions between p53-DBD and DNA can be frequently related to cancer.

The role of the half-turn in determining structures of Alzheimer's Aß wild-type and mutants.

Half-turns are shown to be the main determinants of many experimental Alzheimer's Aß fibril structures. Fibril structures contain three half-turn types, ßαRß, ßαLß and ß뵧 which each result in a ∼90° bend in a ß-strand. It is shown that only these half-turns enable cross-ß stacking and thus the right-angle fold seen in fibrils is an intrinsic feature of cross-ß. Encoding a strand as a conformational sequence in ß, αR, αL and ε(ßL), pairwise combination rules for consecutive half-turns are used to decode this sequence to give the backbone path. This reveals how structures would be dramatically affected by a deletion. Using a wild-type Aß(42) fibril structure and the pairwise combination rules, the Osaka deletion is predicted to result in exposure of surfaces that are mutually shielding from the solvent. Molecular dynamics simulations on an 11-mer ß-sheet of Alzheimer's Aß(40) of the Dutch (E22Q), Iowa (D23N), Arctic (E22G), and Osaka (E22Δ) mutants, show the crucial role glycine plays in the positioning of ßαRß half-turns. Their "in-phase" positions along the sequence in the wild-type, Dutch mutant and Iowa mutant means that the half-folds all fold to the same side creating the same closed structure. Their out-of-phase positions in Arctic and Osaka mutants creates a flatter structure in the former and an S-shape structure in the latter which, as predicted, exposes surfaces on the inside in the closed wild-type to the outside. This is consistent with the gain of interaction model and indicates how domain swapping might explain the Osaka mutant's unique properties.

Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.

Thioredoxin (Trx) is a redox-responsive protein that modulates the activities of its target proteins mostly by reducing their disulfide bonds. In chloroplasts, five Trx isoforms (Trx-f, Trx-m, Trx-x, Trx-y, and Trx-z) regulate various photosynthesis-related enzymes with distinct target selectivity. To elucidate the determinants of the target selectivity of each Trx isoform, here we investigated the residues responsible for target recognition by Trx-f, the most well-studied chloroplast-resident Trx. As reported previously, we found that positively-charged residues on the Trx-f surface are involved in the interactions with its targets. Moreover, several residues that are specifically conserved in Trx-f (e.g. Cys-126 and Thr-158) were also involved in interactions with target proteins. The validity of these residues was examined by the molecular dynamics simulation. In addition, we validated the impact of these key residues on target protein reduction by studying (i) Trx-m variants into which we introduced the key residues for Trx-f and (ii) Trx-like proteins, named atypical Cys His-rich Trx 1 (ACHT1) and ACHT2a, that also contain these key residues. These artificial or natural protein variants could reduce Trx-f-specific targets, indicating that the key residues for Trx-f are critical for Trx-f-specific target recognition. Furthermore, we demonstrate that ACHT1 and ACHT2a efficiently oxidize some Trx-f-specific targets, suggesting that its target selectivity also contributes to the oxidative regulation process. Our results reveal the key residues for Trx-f-specific target recognition and uncover ACHT1 and ACHT2a as oxidation factors of their target proteins, providing critical insight into redox regulation of photosynthesis.

Edge expansion parallel cascade selection molecular dynamics simulation for investigating large-amplitude collective motions of proteins.

We propose edge expansion parallel cascade selection molecular dynamics (eePaCS-MD) as an efficient adaptive conformational sampling method to investigate the large-amplitude motions of proteins without prior knowledge of the conformational transitions. In this method, multiple independent MD simulations are iteratively conducted from initial structures randomly selected from the vertices of a multi-dimensional principal component subspace. This subspace is defined by an ensemble of protein conformations sampled during previous cycles of eePaCS-MD. The edges and vertices of the conformational subspace are determined by solving the "convex hull problem." The sampling efficiency of eePaCS-MD is achieved by intensively repeating MD simulations from the vertex structures, which increases the probability of rare event occurrence to explore new large-amplitude collective motions. The conformational sampling efficiency of eePaCS-MD was assessed by investigating the open-close transitions of glutamine binding protein, maltose/maltodextrin binding protein, and adenylate kinase and comparing the results to those obtained using related methods. In all cases, the open-close transitions were simulated in ∼10 ns of simulation time or less, offering 1-3 orders of magnitude shorter simulation time compared to conventional MD. Furthermore, we show that the combination of eePaCS-MD and accelerated MD can further enhance conformational sampling efficiency, which reduced the total computational cost of observing the open-close transitions by at most 36%.

Universality and Structural Implications of the Boson Peak in Proteins.

The softness and rigidity of proteins are reflected in the structural dynamics, which are in turn affected by the environment. The characteristic low-frequency vibrational spectrum of a protein, known as boson peak, is an indication of the structural rigidity of the protein at a cryogenic temperature or dehydrated conditions. In this article, the effect of hydration, temperature, and pressure on the boson peak and volumetric properties of a globular protein are evaluated by using inelastic neutron scattering and molecular dynamics simulation. Hydration, pressurization, and cooling shift the boson peak position to higher energy and depress the peak intensity and decreases the protein and cavity volumes. We found the correlation between the boson peak and cavity volume in a protein. A decrease of cavity volume means the increase of rigidity, which is the origin of the boson peak shift. Boson peak is the universal property of a protein, which is rationalized by the correlation.

Haptic-Assisted Interactive Molecular Docking Incorporating Receptor Flexibility.

Haptic-assisted interactive docking tools immerse the user in an environment where intuition and knowledge can be used to help guide the docking process. Here we present such a tool where the user "holds" a rigid ligand via a haptic device through which they feel interaction forces with a flexible receptor biomolecule. To ensure forces transmitted through the haptic device are smooth and stable, they must be updated at a rate greater than 500 Hz. Due to this time constraint, the majority of haptic docking tools do not attempt to model the conformational changes that would occur when molecules interact during binding. Our haptic-assisted docking tool, "Haptimol FlexiDock", models a receptor's conformational response to forces of interaction with a ligand while maintaining the required haptic refresh rate. In order to model receptor flexibility we use the method of linear response for which we determine the variance-covariance matrix of atomic fluctuations from the trajectory of an explicit-solvent molecular dynamics simulation of the ligand-free receptor molecule. The key to satisfying the time constraint is an eigenvector decomposition of the variance-covariance matrix which enables a good approximation to the conformational response of the receptor to be calculated rapidly. This exploits a feature of protein dynamics whereby most fluctuation occurs within a relatively small subspace. The method is demonstrated on glutamine binding protein in interaction with glutamine and maltose binding protein in interaction with maltose. For both proteins the movement that occurs when the ligand is docked near to its binding site matches the experimentally determined movement well. It is thought that this tool will be particularly useful for structure-based drug design.

evERdock BAI: Machine-learning-guided selection of protein-protein complex structure.

Computational techniques for accurate and efficient prediction of protein-protein complex structures are widely used for elucidating protein-protein interactions, which play important roles in biological systems. Recently, it has been reported that selecting a structure similar to the native structure among generated structure candidates (decoys) is possible by calculating binding free energies of the decoys based on all-atom molecular dynamics (MD) simulations with explicit solvent and the solution theory in the energy representation, which is called evERdock. A recent version of evERdock achieves a higher-accuracy decoy selection by introducing MD relaxation and multiple MD simulations/energy calculations; however, huge computational cost is required. In this paper, we propose an efficient decoy selection method using evERdock and the best arm identification (BAI) framework, which is one of the techniques of reinforcement learning. The BAI framework realizes an efficient selection by suppressing calculations for nonpromising decoys and preferentially calculating for the promising ones. We evaluate the performance of the proposed method for decoy selection problems of three protein-protein complex systems. Their results show that computational costs are successfully reduced by a factor of 4.05 (in the best case) compared to a standard decoy selection approach without sacrificing accuracy.

Origins of biological function in DNA and RNA hairpin loop motifs from replica exchange molecular dynamics simulation.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have remarkably similar chemical structures, but despite this, they play significantly different roles in modern biology. In this article, we explore the possible conformations of DNA and RNA hairpins to better understand the fundamental differences in structure formation and stability. We use large parallel temperature replica exchange molecular dynamics ensembles to sample the full conformational landscape of these hairpin molecules so that we can identify the stable structures formed by the hairpin sequence. Our simulations show RNA adopts a narrower distribution of folded structures compared to DNA at room temperature, which forms both hairpins and many unfolded conformations. RNA is capable of forming twice as many hydrogen bonds than DNA which results in a higher melting temperature. We see that local chemical differences lead to emergent molecular properties such as increased persistence length in RNA that is weakly temperature dependant. These discoveries provide fundamental insight into how RNA forms complex folded tertiary structures which confer enzymatic-like function in ribozymes, whereas DNA retains structural motifs in order to facilitate function such as translation of sequence.

Binding free energy analysis of protein-protein docking model structures by evERdock.

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Refining evERdock: Improved selection of good protein-protein complex models achieved by MD optimization and use of multiple conformations.

A method for evaluating binding free energy differences of protein-protein complex structures generated by protein docking was recently developed by some of us. The method, termed evERdock, combined short (2 ns) molecular dynamics (MD) simulations in explicit water and solution theory in the energy representation (ER) and succeeded in selecting the near-native complex structures from a set of decoys. In the current work, we performed longer (up to 100 ns) MD simulations before employing ER analysis in order to further refine the structures of the decoy set with improved binding free energies. Moreover, we estimated the binding free energies for each complex structure based on an average value from five individual MD snapshots. After MD simulations, all decoys exhibit a decrease in binding free energy, suggesting that proper equilibration in explicit solvent resulted in more favourably bound complexes. During the MD simulations, non-native structures tend to become unstable and in some cases dissociate, while near-native structures maintain a stable interface. The energies after the MD simulations show an improved correlation between similarity criteria (such as interface root-mean-square distance) to the native (crystal) structure and the binding free energy. In addition, calculated binding free energies show sensitivity to the number of contacts, which was demonstrated to reflect the relative stability of structures at earlier stages of the MD simulation. We therefore conclude that the additional equilibration step along with the use of multiple conformations can make the evERdock scheme more versatile under low computational cost.

Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor.

The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H3O(+) permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor.

Functional Conversion of CPD and (6-4) Photolyases by Mutation.

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.

Design, synthesis and structure-activity relationship studies of novel sirtuin 2 (SIRT2) inhibitors with a benzamide skeleton.

Human sirtuin 2 (SIRT2) is an attractive target molecule for development of drugs to treat neurodegenerative diseases and cancer, because SIRT2 inhibitors have a protective effect against neurodegeneration and an anti-proliferative effect on cancer stem cells. We designed and synthesized a series of benzamide derivatives as SIRT2 inhibitor candidates. Among them, compound 17k showed the most potent SIRT2-inhibitory activity (IC50=0.60µM), with more than 150-fold selectivity over SIRT1 and SIRT3 isoforms (IC50 >100µM).

Ligand-induced protein responses and mechanical signal propagation described by linear response theories.

In this study, a general linear response theory (LRT) is formulated to describe time-dependent and -independent protein conformational changes upon CO binding with myoglobin. Using the theory, we are able to monitor protein relaxation in two stages. The slower relaxation is found to occur from 4.4 to 81.2 picoseconds and the time constants characterized for a couple of aromatic residues agree with those observed by UV Resonance Raman (UVRR) spectrometry and time resolved x-ray crystallography. The faster "early responses", triggered as early as 400 femtoseconds, can be best described by the theory when impulse forces are used. The newly formulated theory describes the mechanical propagation following ligand-binding as a function of time, space and types of the perturbation forces. The "disseminators", defined as the residues that propagate signals throughout the molecule the fastest among all the residues in protein when perturbed, are found evolutionarily conserved and the mutations of which have been shown to largely change the CO rebinding kinetics in myoglobin.

PaCS-Toolkit: Optimized Software Utilities for Parallel Cascade Selection Molecular Dynamics (PaCS-MD) Simulations and Subsequent Analyses.

Parallel cascade selection molecular dynamics (PaCS-MD) is an enhanced conformational sampling method conducted as a "repetition of time leaps in parallel worlds", comprising cycles of multiple molecular dynamics (MD) simulations performed in parallel and selection of the initial structures of MDs for the next cycle. We developed PaCS-Toolkit, an optimized software utility enabling the use of different MD software and trajectory analysis tools to facilitate the execution of the PaCS-MD simulation and analyze the obtained trajectories, including the preparation for the subsequent construction of the Markov state model. PaCS-Toolkit is coded with Python, is compatible with various computing environments, and allows for easy customization by editing the configuration file and specifying the MD software and analysis tools to be used. We present the software design of PaCS-Toolkit and demonstrate applications of PaCS-MD variations: original targeted PaCS-MD to peptide folding; rmsdPaCS-MD to protein domain motion; and dissociation PaCS-MD to ligand dissociation from adenosine A2A receptor.

CyClus: a fast, comprehensive cylindrical interface approximation clustering/reranking method for rigid-body protein-protein docking decoys.

We propose a fast clustering and reranking method, CyClus, for protein-protein docking decoys. This method enables comprehensive clustering of whole decoys generated by rigid-body docking using cylindrical approximation of the protein-proteininterface and hierarchical clustering procedures. We demonstrate the clustering and reranking of 54,000 decoy structures generated by ZDOCK for each complex within a few minutes. After parameter tuning for the test set in ZDOCK benchmark 2.0 with the ZDOCK and ZRANK scoring functions, blind tests for the incremental data in ZDOCK benchmark 3.0 and 4.0 were conducted. CyClus successfully generated smaller subsets of decoys containing near-native decoys. For example, the number of decoys required to create subsets containing near-native decoys with 80% probability was reduced from 22% to 50% of the number required in the original ZDOCK. Although specific ZDOCK and ZRANK results were demonstrated, the CyClus algorithm was designed to be more general and can be applied to a wide range of decoys and scoring functions by adjusting just two parameters, p and T. CyClus results were also compared to those from ClusPro.