PoSSuM v.3: A Major Expansion of the PoSSuM Database for Finding Similar Binding Sites of Proteins.

Information on structures of protein-ligand complexes, including comparisons of known and putative protein-ligand-binding pockets, is valuable for protein annotation and drug discovery and development. To facilitate biomedical and pharmaceutical research, we developed PoSSuM (https://possum.cbrc.pj.aist.go.jp/PoSSuM/), a database for identifying similar binding pockets in proteins. The current PoSSuM database includes 191 million similar pairs among almost 10 million identified pockets. PoSSuM drug search (PoSSuMds) is a resource for investigating ligand and receptor diversity among a set of pockets that can bind to an approved drug compound. The enhanced PoSSuMds covers pockets associated with both approved drugs and drug candidates in clinical trials from the latest release of ChEMBL. Additionally, we developed two new databases: PoSSuMAg for investigating antibody-antigen interactions and PoSSuMAF to simplify exploring putative pockets in AlphaFold human protein models.

A multipoint guidance mechanism for ß-barrel folding on the SAM complex.

Mitochondrial ß-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic ß-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last ß-strand (ß-signal) of the substrate-the 19-ß-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first ß-segment (ß1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces ß1 to promote its pairing with Tom40's last ß-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial ß-barrel folding.

Protein-protein interaction prediction methods: from docking-based to AI-based approaches.

Protein-protein interactions (PPIs), such as protein-protein inhibitor, antibody-antigen complex, and supercomplexes play diverse and important roles in cells. Recent advances in structural analysis methods, including cryo-EM, for the determination of protein complex structures are remarkable. Nevertheless, much room remains for improvement and utilization of computational methods to predict PPIs because of the large number and great diversity of unresolved complex structures. This review introduces a wide array of computational methods, including our own, for estimating PPIs including antibody-antigen interactions, offering both historical and forward-looking perspectives.

Application of Homology Modeling by Enhanced Profile-Profile Alignment and Flexible-Fitting Simulation to Cryo-EM Based Structure Determination.

Application of cryo-electron microscopy (cryo-EM) is crucially important for ascertaining the atomic structure of large biomolecules such as ribosomes and protein complexes in membranes. Advances in cryo-EM technology and software have made it possible to obtain data with near-atomic resolution, but the method is still often capable of producing only a density map with up to medium resolution, either partially or entirely. Therefore, bridging the gap separating the density map and the atomic model is necessary. Herein, we propose a methodology for constructing atomic structure models based on cryo-EM maps with low-to-medium resolution. The method is a combination of sensitive and accurate homology modeling using our profile-profile alignment method with a flexible-fitting method using molecular dynamics simulation. As described herein, this study used benchmark applications to evaluate the model constructions of human two-pore channel 2 (one target protein in CASP13 with its structure determined using cryo-EM data) and the overall structure of Enterococcus hirae V-ATPase complex.

Crystal structure of Tam41 cytidine diphosphate diacylglycerol synthase from a Firmicutes bacterium.

Translocator assembly and maintenance 41 (Tam41) catalyses the synthesis of cytidine diphosphate diacylglycerol (CDP-DAG), which is a high-energy intermediate phospholipid critical for generating cardiolipin in mitochondria. Although Tam41 is present almost exclusively in eukaryotic cells, a Firmicutes bacterium contains the gene encoding Tam41-type CDP-DAG synthase (FbTam41). FbTam41 converted phosphatidic acid (PA) to CDP-DAG using a ternary complex mechanism in vitro. Additionally, FbTam41 functionally substituted yeast Tam41 in vivo. These results demonstrate that Tam41-type CDP-DAG synthase functions in some prokaryotic cells. We determined the crystal structure of FbTam41 lacking the C-terminal 18 residues in the cytidine triphosphate (CTP)-Mg2+ bound form at a resolution of 2.6 Å. The crystal structure showed that FbTam41 contained a positively charged pocket that specifically accommodated CTP-Mg2+ and PA in close proximity. By using this structure, we constructed a model for the full-length structure of FbTam41 containing the last a-helix, which was missing in the crystal structure. Based on this model, we propose a molecular mechanism for CDP-DAG synthesis in bacterial cells and mitochondria.

HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer's disease pathology.

DNA damage is increased in Alzheimer's disease (AD), while the underlying mechanisms are unknown. Here, we employ comprehensive phosphoproteome analysis, and identify abnormal phosphorylation of 70 kDa subunit of Ku antigen (Ku70) at Ser77/78, which prevents Ku70-DNA interaction, in human AD postmortem brains. The abnormal phosphorylation inhibits accumulation of Ku70 to the foci of DNA double strand break (DSB), impairs DNA damage repair and eventually causes transcriptional repression-induced atypical cell death (TRIAD). Cells under TRIAD necrosis reveal senescence phenotypes. Extracellular high mobility group box 1 (HMGB1) protein, which is released from necrotic or hyper-activated neurons in AD, binds to toll-like receptor 4 (TLR4) of neighboring neurons, and activates protein kinase C alpha (PKCα) that executes Ku70 phosphorylation at Ser77/78. Administration of human monoclonal anti-HMGB1 antibody to post-symptomatic AD model mice decreases neuronal DSBs, suppresses secondary TRIAD necrosis of neurons, prevents escalation of neurodegeneration, and ameliorates cognitive symptoms. TRIAD shares multiple features with senescence. These results discover the HMGB1-Ku70 axis that accounts for the increase of neuronal DNA damage and secondary enhancement of TRIAD, the cell death phenotype of senescence, in AD.

Mitochondrial sorting and assembly machinery operates by ß-barrel switching.

The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.

Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions.

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-ß, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.

Free-energy analysis of the hydration and cosolvent effects on the ß-sheet aggregation through all-atom molecular dynamics simulation.

Energetics was analyzed for the aggregation of an 11-residue peptide. An all-atom molecular dynamics simulation was conducted with explicit solvent, and the energy-representation theory of solution was employed to compute the solvation free energies of the peptide and its aggregates. The aggregation in the pure-water solvent was observed to be inhibited by the solvation. The driving force of aggregate formation is the interactions among the peptide molecules, and the sum of the intra-aggregate and solvation terms per monomer is more favorable for larger aggregates. The effect of the cosolvent was then examined by focusing on the mixtures of water with urea and dimethyl sulfoxide (DMSO). It was actually shown that the derivative of the excess chemical potential of a flexible solute species with respect to the cosolvent concentration is determined exactly by the corresponding derivative of the free energy of solvation. The cosolvent effect on the equilibrium of aggregate formation can thus be addressed by comparing the solvation free energies with and without the cosolvent, and both the urea and DMSO cosolvents were found to inhibit the aggregation. The cosolvent-induced change in the solvation free energy was further decomposed into the contributions from the cosolvent and water. Their dependencies on the degree of aggregation were seen to be weak for large aggregates, and the roles of the electrostatic, van der Waals, and excluded-volume components in the solvation energetics were discussed.

Probabilistic analysis for identifying the driving force of protein folding.

Toward identifying the driving force of protein folding, energetics was analyzed in water for Trp-cage (20 residues), protein G (56 residues), and ubiquitin (76 residues) at their native (folded) and heat-denatured (unfolded) states. All-atom molecular dynamics simulation was conducted, and the hydration effect was quantified by the solvation free energy. The free-energy calculation was done by employing the solution theory in the energy representation, and it was seen that the sum of the protein intramolecular (structural) energy and the solvation free energy is more favorable for a folded structure than for an unfolded one generated by heat. Probabilistic arguments were then developed to determine which of the electrostatic, van der Waals, and excluded-volume components of the interactions in the protein-water system governs the relative stabilities between the folded and unfolded structures. It was found that the electrostatic interaction does not correspond to the preference order of the two structures. The van der Waals and excluded-volume components were shown, on the other hand, to provide the right order of preference at probabilities of almost unity, and it is argued that a useful modeling of protein folding is possible on the basis of the excluded-volume effect.

Interaction-component analysis of the effects of urea and its alkylated derivatives on the structure of T4-lysozyme.

The effects of urea and its alkylated derivatives on the structure of T4-lysozyme were analyzed from the standpoint of energetics. Molecular dynamics simulations were conducted with explicit solvent, and the energy-representation method was employed to compute the free energy of transfer of the protein from pure-water solvent to the mixed solvents of water with urea, methylurea, 1,1-dimethylurea, and isopropylurea. Through the decomposition of the transfer free energy into the cosolvent and water contributions, it was observed that the former is partially cancelled by the latter and governs the total free energy of transfer. To determine the interaction component responsible for the transfer energetics, the correlations of the transfer free energy were also examined against the change in the solute-solvent interaction energy upon transfer and the corresponding changes in the electrostatic, van der Waals, and excluded-volume components. It was then found over the set of protein structures ranging from native to (partially) unfolded ones that the transfer free energy changes in parallel with the van der Waals component even when the cosolvent is alkylated. The electrostatic and excluded-volume components play minor roles in the structure modification of the protein, and the denaturing ability of alkylurea is brought by the van der Waals interaction.

Interaction-component analysis of the hydration and urea effects on cytochrome c.

Energetics was analyzed for cytochrome c in pure-water solvent and in a urea-water mixed solvent to elucidate the solvation effect in the structural variation of the protein. The solvation free energy was computed through all-atom molecular dynamics simulation combined with the solution theory in the energy representation, and its correlations were examined over sets of protein structures against the electrostatic and van der Waals components in the average interaction energy of the protein with the solvent and the excluded-volume component in the solvation free energy. It was observed in pure-water solvent that the solvation free energy varies in parallel to the electrostatic component with minor roles played by the van der Waals and excluded-volume components. The effect of urea on protein structure was then investigated in terms of the free-energy change upon transfer of the protein solute from pure-water solvent to the urea-water mixed solvent. The decomposition of the transfer free energy into the contributions from urea and water showed that the urea contribution is partially canceled by the water contribution and governs the total free energy of transfer. When correlated against the change in the solute-solvent interaction energy upon transfer and the corresponding changes in the electrostatic, van der Waals, and excluded-volume components, the transfer free energy exhibited strong correlations with the total change in the solute-solvent energy and its van der Waals component. The solute-solvent energy was decomposed into the contributions from the protein backbone and side chain, furthermore, and neither of the contributions was seen to be decisive in the correlation to the transfer free energy.

MuSTAR MD: multi-scale sampling using temperature accelerated and replica exchange molecular dynamics.

A new and efficient conformational sampling method, MuSTAR MD (Multi-scale Sampling using Temperature Accelerated and Replica exchange Molecular Dynamics), is proposed to calculate the free energy landscape on a space spanned by a set of collective variables. This method is an extension of temperature accelerated molecular dynamics and can also be considered as a variation of replica-exchange umbrella sampling. In the MuSTAR MD, each replica contains an all-atom fine-grained model, at least one coarse-grained model, and a model defined by the collective variables that interacts with the other models in the same replica through coupling energy terms. The coarse-grained model is introduced to drive efficient sampling of large conformational space and the fine-grained model can serve to conduct more accurate conformational sampling. The collective variable model serves not only to mediate the coarse- and fine-grained models, but also to enhance sampling efficiency by temperature acceleration. We have applied this method to Ala-dipeptide and examined the sampling efficiency of MuSTAR MD in the free energy landscape calculation compared to that for replica exchange molecular dynamics, replica exchange umbrella sampling, temperature accelerated molecular dynamics, and conventional MD. The results clearly indicate the advantage of sampling a relatively high energy conformational space, which is not sufficiently sampled with other methods. This feature is important in the investigation of transition pathways that go across energy barriers. MuSTAR MD was also applied to Met-enkephalin as a test case in which two Go-like models were employed as the coarse-grained model.