Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

Free-Energy Profiles for Membrane Permeation of Compounds Calculated Using Rare-Event Sampling Methods.

The free-energy profile of a compound is an essential measurement in evaluating the membrane permeation process by means of theoretical methods. Computationally, molecular dynamics (MD) simulation allows the free-energy profile calculation. However, MD simulations frequently fail to sample membrane permeation because they are rare events induced in longer timescales than the accessible timescale of MD, leading to an insufficient conformational search to calculate an incorrect free-energy profile. To achieve a sufficient conformational search, several enhanced sampling methods have been developed and elucidated the membrane permeation process. In addition to these enhanced sampling methods, we proposed a simple yet powerful free-energy calculation of a compound for the membrane permeation process based on originally rare-event sampling methods developed by us. Our methods have a weak dependency on external biases and their optimizations to promote the membrane permeation process. Based on distributed computing, our methods only require the selection of initial structures and their conformational resampling, whereas the enhanced sampling methods may be required to adjust external biases. Furthermore, our methods efficiently search membrane permeation processes with simple scripts without modifying any MD program. As demonstrations, we calculated the free-energy profiles of seven linear compounds for their membrane permeation based on a hybrid conformational search using two rare-event sampling methods, that is, (1) parallel cascade selection MD (PaCS-MD) and (2) outlier flooding method (OFLOOD), combined with a Markov state model (MSM) construction. In the first step, PaCS-MD generated initial membrane permeation paths of a compound. In the second step, OFLOOD expanded the unsearched conformational area around the initial paths, allowing for a broad conformational search. Finally, the trajectories were employed to construct reliable MSMs, enabling correct free-energy profile calculations. Furthermore, we estimated the membrane permeability coefficients of all compounds by constructing the reliable MSMs for their membrane permeation. In conclusion, the calculated coefficients were qualitatively correlated with the experimental measurements (correlation coefficient (R2) = 0.8689), indicating that the hybrid conformational search successfully calculated the free-energy profiles and membrane permeability coefficients of the seven compounds.

Efficient screening of protein-ligand complexes in lipid bilayers using LoCoMock score.

Membrane proteins are attractive targets for drug discovery due to their crucial roles in various biological processes. Studying the binding poses of amphipathic molecules to membrane proteins is essential for understanding the functions of membrane proteins and docking simulations can facilitate the screening of protein-ligand complexes at low computational costs. However, identifying docking poses for a ligand in non-aqueous environments such as lipid bilayers can be challenging. To address this issue, we propose a new docking score called logP-corrected membrane docking (LoCoMock) score. To screen putative protein-ligand complexes embedded in a membrane, the LoCoMock score considers the affinity between a target ligand and the membrane. It combines the docking score of the protein-ligand complex with the logP of the target ligand. In demonstrations using several model ligands, the LoCoMock score screened more putative complexes than the conventional docking score. As extended docking, the LoCoMock score makes it possible to screen membrane proteins more effectively as drug targets than the conventional docking.

Phosphorylation in the accessory domain of yeast histone chaperone protein 1 exposes the nuclear export signal sequence.

Histone chaperone proteins assist in the formation of the histone octamers, the scaffold proteins that facilitate the packing of DNA into nucleosomes in the cell nucleus. One such histone chaperone protein is yeast nucleosome assembly protein 1 (yNap1), the crystal structure of which has been determined and found to have a nuclear export signal (NES) sequence within its long α-helix. Experimental evidence obtained from mutagenesis studies of the budding yeast suggests that the NES is necessary for the transport of yNap1 from the cell nucleus to the cytosol. However, the NES sequence is masked by an accessory domain, the exact role of which has not yet been elucidated, especially in nucleocytoplasmic transport. To clarify the role of the accessory domain, we focused on its phosphorylation, because proteomic experiments have identified multiple phosphorylation sites on yNap1. To study this phenomenon computationally, all-atom molecular dynamics simulations of the non-phosphorylated yNap1 (Nap1-nonP) and phosphorylated yNap1 (Nap1-P) systems were performed. Specifically, we addressed how the NES sequence is exposed to the protein surface by measuring its solvent-accessible surface area (SASA). It was found that the median of the SASA distribution of Nap1-P was greater than that of Nap1-nonP, indicating that phosphorylation in the accessory domain exposes the NES, resulting in its increased accessibility. In conclusion, yNap1 might modulate the accessibility of the NES by dislocating the accessory domain through its phosphorylation.

Structural Validation by the G-Factor Properly Regulates Boost Potentials Imposed in Conformational Sampling of Proteins.

Free energy landscapes (FELs) of proteins are indispensable for evaluating thermodynamic properties. Molecular dynamics (MD) simulation is a computational method for calculating FELs; however, conventional MD simulation frequently fails to search a broad conformational subspace due to its accessible timescale, which results in the calculation of an unreliable FEL. To search a broad subspace, an external bias can be imposed on a protein system, and biased sampling tends to cause a strong perturbation that might collapse the protein structures, indicating that the strength of the external bias should be properly regulated. This regulation can be challenging, and empirical parameters are frequently employed to impose an optimal bias. To address this issue, several methods regulate the external bias by referring to system energies. Herein, we focused on protein structural information for this regulation. In this study, a well-established structural indicator (the G-factor) was used to obtain structural information. Based on the G-factor, we proposed a scheme for regulating biased sampling, which is referred to as a G-factor-based external bias limiter (GERBIL). With GERBIL, the configurations were structurally validated by the G-factor during biased sampling. As an example of biased sampling, an accelerated MD (aMD) simulation was adopted in GERBIL (aMD-GERBIL), whereby the aMD simulation was repeatedly performed by increasing the strength of the boost potential. Furthermore, the configurations sampled by the aMD simulation were structurally validated by their G-factor values, and aMD-GERBIL stopped increasing the strength of the boost potential when the sampled configurations were regarded as low-quality (collapsed) structures. This structural validation is regarded as a "Brake" of the boost potential. For demonstrations, aMD-GERBIL was applied to globular proteins (ribose binding and maltose-binding proteins) to promote their large-amplitude open-closed transitions and successfully identify their domain motions.

Protein Structure Validation Derives a Smart Conformational Search in a Physically Relevant Configurational Subspace.

Since proteins perform biological functions through their dynamic properties, molecular dynamics (MD) simulation is a sophisticated strategy for investigating their functions. Analyses of trajectories provide statistical information about a specific protein as a free-energy landscape (FEL). However, the timescale of normal MD is shorter than that of biological functions, resulting in statistically insufficient conformational sampling, finally leading to unreliable FEL calculation. To search for a broad configurational subspace, an external bias is imposed on a target protein as biased sampling. However, its regulation is challenging because the optimal strength of the perturbation is unknown. Furthermore, a physically irrelevant configurational subspace was searched when imposing an inappropriate external bias. To address this issue, we newly proposed an external biased regulation scheme known as the G-factor external bias limiter (GERBIL). In GERBIL, protein configurations generated by external bias are structurally validated by an indicator (G-factor), enabling the search for a physically relevant subspace. In addition to biased sampling, nonbiased sampling might search for a physically irrelevant configurational subspace because repeating multiple MD simulations from several initial structures tends to search for an overly broad configurational subspace. For this issue, the structural qualities of configurations generated by nonbiased sampling have not been investigated. Therefore, we confirmed whether the G-factor screened the collapsed (low-quality) configurations generated by nonbiased sampling. To address this issue, the outlier flooding method (OFLOOD) was adopted in GERBIL as a nonbiased sampling method, which is referred to as OFLOOD-GERBIL. OFLOOD rapidly expands a configurational subspace by resampling the rarely occurring states of a given protein and tends to search an overly broad subspace. Thus, we considered that GERBIL might improve the excessive conformational search of OFLOOD for a physically irrelevant configurational subspace. As a demonstration, OFLOOD and OFLOOD-GERBIL were applied to a globular protein (T4 lysozyme) and their conformational search qualities were assessed. Based on our assessment, normal OFLOOD without the outlier validation frequently sampled low-quality configurations, whereas OFLOOD-GERBIL with the outlier validation intensively sampled high-quality configurations. In conclusion, OFLOOD-GERBIL derives a smart conformational search in a physically relevant configurational subspace, indicating that protein structure validation works in both nonbiased and biased sampling methods.

Comprehensive predictions of secondary structures for comparative analysis in different species.

Protein structures are directly linked to biological functions. However, there is a gap of knowledge between the decoded genome and the structure. To bridge the gap, we focused on the secondary structure (SS). From a comprehensive analysis of predicted SS of proteins in different types of organisms, we have arrived at the following findings: The proportions of SS in genomes were different among phylogenic domains. The distributions of strand lengths indicated structural limitations in all of the species. Different from bacteria and archaea, eukaryotes have an abundance of α-helical and random coil proteins. Interestingly, there was a relationship between SS and post-translational modifications. By calculating hydrophobicity moments of helices and strands, highly amphipathic fragments of SS were found, which might be related to the biological functions. In conclusion, comprehensive predictions of SS will provide valuable perspectives to understand the entire protein structures in genomes and will help one to discover or design functional proteins.

Cell cycle-dependent phosphorylation of IQGAP is involved in assembly and stability of the contractile ring in fission yeast.

Cytokinesis is the final step in cell division and is driven by the constriction of the medial actomyosin-based contractile ring (CR) in many eukaryotic cells. In the fission yeast Schizosaccharomyces pombe, the IQGAP-like protein Rng2 is required for assembly and constriction of the CR, and specifically interacts with actin filaments (F-actin) in the CR after anaphase. However, the mechanism that timely activates Rng2 has not yet been elucidated. We herein tested the hypothesis that the cytokinetic function of Rng2 is regulated by phosphorylation by examining phenotypes of a series of non-phosphorylatable and phosphomimetic rng2 mutant strains. In phosphomimetic mutant cells, F-actin in the CR was unstable. Genetic analyses indicated that phosphorylated Rng2 was involved in CR assembly in cooperation with myosin-II, whereas the phosphomimetic mutation attenuated the localization of Rng2 to CR F-actin. The present results suggest that Rng2 is phosphorylated during CR assembly and then dephosphorylated, which enhances the interaction between Rng2 and CR F-actin to stabilize the ring, thereby ensuring secure cytokinesis.

In silico mutational analyses reveal different ligand-binding abilities of double pockets of medaka fish taste receptor type 1 essential for efficient taste recognition.

Taste receptors are important sensors for the detection of nutrient concentrations in animals. Tastes are recognized by interactions between chemical substances and taste receptors. Recently, the high-resolution X-ray crystal structure of the extracellular ligand-binding domains (LBDs) of medaka fish (Oryzias latipes) taste receptor type 1 (T1r) complexed with ligands (amino acids) was determined. Medaka fish T1r is a heterodimer composed of two different LBDs, T1r2aLBD and T1r3LBD. In this study, we performed all-atom molecular dynamics (MD) simulations on this heterodimer (T1r2aLBD-T1r3LBD) to address mutational effects on key residues near each ligand-binding pocket in recognizing one of the ligands (L-Gln). For T1r2aLBD, Ser165 is important in ligand recognition due to its direct hydrogen bonding with the ligand. After mutating Ser165 to Ile or Ala, the direct hydrogen bonds between the ligand and the binding pocket were weakened, which destabilized the ligand-binding form of T1r2aLBD. For T1r3LBD, Ser300 is important in ligand recognition. The water-mediated hydrogen bond with the side-chain hydroxyl group of Ser300 is a single interaction that maintains the ligand-binding form of T1r3LBD. After mutating Ser300 to Glu or Ala, both mutant systems almost maintained their ligand-binding form. As a mechanism for maintaining the binding form of T1r3LBD, alternative hydrogen bonds were formed as direct interactions instead of the indirect water-mediated interactions found in the wild-type system, which stabilized the binding form of T1r3LBD. Judging from our in silico mutational analyses, T1r2aLBD was structurally destabilized by the amino acid mutations. Therefore, it might be required that the ligand-binding pocket of T1r2aLBD is composed of a set of specific residues to maintain its ligand-binding form. On the contrary, T1r3LBD was robust enough to withstand the amino acid mutations. These different ligand-binding abilities of both LBDs provide multiple binding modes, which might be helpful for discriminating various taste substances or detecting concentrations of nutrients efficiently.

Latrunculin resistance mechanism of non-conventional actin NAP1 uncovered by molecular dynamics simulations.

Monomeric G-actin polymerizes into F-actin to perform various cellular functions. Actin depolymerization drugs, such as latrunculin-A (Lat-A), inhibit filament formation and disrupt the cytoskeleton. Interestingly, the green algae Chlamydomonas alternatively produces a non-conventional actin, NAP1, that responds to inhibition by latrunculin. However, the molecular mechanism underlying latrunculin resistance of NAP1 remains unclear because of the difficulty due to its low in vitro polymerizability. Instead of biochemical experiments, we performed molecular dynamics (MD) simulations to investigate whether NAP1 has a lower affinity for Lat-A than the conventional actins. Our phylogenetic comparison of the binding free energies shows that Lat-A is evolutionarily optimized for skeletal muscles. By decomposing the binding free energy into each amino acid residue, we found that some residues in NAP1 play an important role in latrunculin resistance, suggesting that the primary mechanism of latrunculin resistance is the loss of affinity for Lat-A due to substitutions. In conclusion, our binding-free-energy calculations using MD simulations provide the critical insight that loss of affinity is the direct mechanism of latrunculin resistance.

Histone H3 Inhibits Ubiquitin-Ubiquitin Intermolecular Interactions to Enhance Binding to DNA Methyl Transferase 1.

DNA methyltransferase 1 (Dnmt1) is crucial for cell maintenance and preferentially methylates hemimethylated DNA. Recently, a study revealed that Dnmt1 is timely and site-specifically activated by several types of two-mono-ubiquitinated histone H3 tails (H3Ts). However, the molecular mechanism of Dnmt1 activation has not yet been determined, in addition to the role of H3T. Based on experimental data, two-mono-ubiquitinated H3Ts activate Dnmt1 by binding, with different binding affinities. In contrast, ubiquitin molecules unlinked with H3T do not bind to Dnmt1. Despite the existence of experimental data, it is unclear why the binding affinities for Dnmt1 are different. To obtain new insights into the activation mechanism of Dnmt1, we performed all-atom molecular dynamics (MD) simulations on three systems: (1) K14/K18, (2) K14/K23 mono-ubiquitinated H3Ts, and (3) two ubiquitin molecules unlinked with H3T. As an analysis of our MD trajectories, these ubiquitylation patterns modulated ubiquitin-ubiquitin intermolecular interactions. More specifically, the intermolecular contacts between a pair of ubiquitin molecules linked with H3T became weak in the presence of H3T, indicating that H3T makes a cleft between them to inhibit their intermolecular interactions. For these three systems, the intermolecular interactions between the ubiquitin molecules calculated by our MD simulations are in good agreement with the binding affinities for Dnmt1 experimentally measured in a previous study. Therefore, we conclude that H3T acts as a spacer to inhibit ubiquitin-ubiquitin intermolecular interactions, enhancing binding to Dnmt1.

Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability.

Hevin is a secreted extracellular matrix protein that is encoded by the SPARCL1 gene. Recent studies have shown that Hevin plays an important role in regulating synaptogenesis and synaptic plasticity. Mutations in the SPARCL1 gene increase the risk of autism spectrum disorder (ASD). However, the molecular basis of how mutations in SPARCL1 increase the risk of ASD is not been fully understood. In this study, we show that one of the SPARCL1 mutations associated with ASD impairs normal Hevin secretion. We identified Hevin mutants lacking the EF-hand motif through analyzing ASD-related mice with vulnerable spliceosome functions. Hevin deletion mutants accumulate in the endoplasmic reticulum (ER), leading to the activation of unfolded protein responses. We also found that a single amino acid substitution of Trp647 with Arg in the EF-hand motif associated with a familial case of ASD causes a similar phenotype in the EF-hand deletion mutant. Importantly, molecular dynamics (MD) simulation revealed that this single amino acid substitution triggers exposure of a hydrophobic amino acid to the surface, increasing the binding of Hevin with molecular chaperons, BIP. Taken together, these data suggest that the integrity of the EF-hand motif in Hevin is crucial for proper folding and that ASD-related mutations impair the export of Hevin from the ER. Our data provide a novel mechanism linking a point mutation in the SPARCL1 gene to the molecular and cellular characteristics involved in ASD.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases.

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Structural Variations of Metallothionein with or without Zinc Ions Elucidated by All-Atom Molecular Dynamics Simulations.

Metallothionein (MT) is a small globular protein that binds to trace metals. However, it was still unclear how the existence of metal ions affects the structure of MT. Therefore, we performed all-atom molecular dynamics (MD) simulations under several surrounding conditions with or without Zn2+ ions. As a result of 10 µs MD simulation, MT without Zn2+ ions tended to adopt an extended ß-hairpin structure, while MT with Zn2+ ions became a globular structure like the NMR structure. Furthermore, we also found that the capture of Zn2+ ions by the second and third cysteines played a crucial role in the formation of the native structure. The finding of the Zn2+ binding for the specific cysteines and the unknown ß-hairpin structure will provide new insights into the structural mechanism of metal signaling.

Independent Nontargeted Parallel Cascade Selection Molecular Dynamics (Ino-PaCS-MD) to Enhance the Conformational Sampling of Proteins.

Biological functions are related to long-time protein dynamics (rare events) that are induced over microseconds. Such protein dynamics can be investigated using molecular dynamics (MD) simulations. However, the detection of rare events remains challenging using conventional MD (cMD) since the accessible timescales of cMD are shorter than those of the biological functions. Recently, the parallel cascade selection MD (PaCS-MD) has been proposed to detect such rare events, wherein transition paths are generated between a given reactant and product. As an extension, the nontargeted PaCS-MD (nt-PaCS-MD) has been proposed to predict the transition paths without requiring reference to any product. Thus, as a further extension, we herein propose independent nt-PaCS-MD, namely, Ino-PaCS-MD, wherein multiple walkers are launched from a set of different starting configurations. Each walker repeats a cycle of restarting short-time MD simulations from configurations with high potentials for making transitions to neighboring metastable states. To further enhance the sampling ability, Ino-PaCS-MD temporarily stops the conformational search and periodically resets the starting configurations so that they are uniformly distributed in a conformational subspace, thereby preventing a given protein from being trapped in one of the metastable states. As a demonstration, Ino-PaCS-MD successfully detects rare events of a maltose-binding protein as open-close transitions with a nanosecond-order simulation time, although a microsecond-order cMD simulation failed to detect these rare events, showing the high sampling efficiency of Ino-PaCS-MD.

Molecular Mechanism for the Actin-Binding Domain of α-Actinin Ain1 Elucidated by Molecular Dynamics Simulations and Mutagenesis Experiments.

In the fission yeast Schizosaccharomyces pombe, α-actinin Ain1 bundles F-actin into the contractile ring (CR) in the middle of the cell. Previous studies have proposed that a conformational change of the actin-binding domain (ABD) of Ain1 enhances the actin-binding activity. However, the molecular mechanism of the conformational change remains to be unveiled at an atomic resolution due to the difficulties of experimental techniques to observe them. In the present study, we performed a set of microsecond-order molecular dynamics (MD) simulations for ABD of Ain1. Our MD simulations for a pathogenic point mutation (R216E) in ABD did not result in large domain motions as previously expected. However, local motions of the loop regions were detected. Besides the three conventional actin-binding sites, we found characteristic electrostatic interactions with the N-terminal of actin. The mutagenesis experiment in fission yeast showed that collapses of the electrostatic interactions at the binding site abolished the proper localization of Ain1 to the CR. Furthermore, the MD simulation of F-actin with the Ain1 ABD R216E indicated that the stronger affinity is caused by a direct interaction of the point mutation. Our findings might be applicable to other highly conserved ABP family proteins to explain their binding affinities.

Molecular dissection of the actin-binding ability of the fission yeast α-actinin, Ain1, in vitro and in vivo.

A contractile ring (CR) is involved in cytokinesis in animal and yeast cells. Although several types of actin-bundling proteins associate with F-actin in the CR, their individual roles in the CR have not yet been elucidated in detail. Ain1 is the sole α-actinin homologue in the fission yeast Schizosaccharomyces pombe and specifically localizes to the CR with a high turnover rate. S. pombe cells lacking the ain1+ gene show defects in cytokinesis under stress conditions. We herein investigated the biochemical activity and cellular localization mechanisms of Ain1. Ain1 showed weaker affinity to F-actin in vitro than other actin-bundling proteins in S. pombe. We identified a mutation that presumably loosened the interaction between two calponin-homology domains constituting the single actin-binding domain (ABD) of Ain1, which strengthened the actin-binding activity of Ain1. This mutant protein induced a deformation in the ring shape of the CR. Neither a truncated protein consisting only of an N-terminal ABD nor a truncated protein lacking a C-terminal region containing an EF-hand motif localized to the CR, whereas the latter was involved in the bundling of F-actin in vitro. We herein propose detailed mechanisms for how each part of the molecule is involved in the proper cellular localization and function of Ain1.