Dual-wield NTPases: A novel protein family mined from AlphaFold DB.

AlphaFold protein structure database (AlphaFold DB) archives a vast number of predicted models. We conducted systematic data mining against AlphaFold DB and discovered an uncharacterized P-loop NTPase family. The structure of the protein family was surprisingly novel, showing an atypical topology for P-loop NTPases, noticeable twofold symmetry, and two pairs of independent putative active sites. Our findings show that structural data mining is a powerful approach to identifying undiscovered protein families.

Elastic network model reveals distinct flexibilities of capping proteins bound to CARMIL and twinfilin-tail.

Capping protein (CP) binds to the barbed end of an actin-filament and inhibits its elongation. CARMIL binds CP and dissociates it from the barbed end of the actin-filament. The binding of CARMIL peptide alters the flexibility of CP, which is considered to facilitate the dissociation. Twinfilin also binds to CP through its C-terminal tail. The complex structures of the CP/twinfilin-tail (TW-tail) peptide indicate that the binding sites of CARMIL and TW-tail overlap. However, TW-tail binding does not facilitate the dissociation of CP from the barbed end. We extensively investigated the flexibilities of CP in the CP/TW-tail or CP/CARMIL complexes using an elastic network model and concluded that TW-tail binding does not alter the flexibility of CP. Our extensive analysis also highlighted that the strong contacts of peptides with the two domains of CP, that is, the CP-L and CP-S domains, are key to changing the flexibilities of CP. CARMIL peptides can interact strongly with both of the domains, while TW-tail peptides exclusively interact with the CP-S domain because the binding site of TW-tail on CP relatively shifts to the CP-S domain compared with that of CP/CARMIL. This result supports our hypothesis that the dissociation of CP from the barbed end is regulated by the flexibility of CP.

Computational Methods to Predict Intrinsically Disordered Regions and Functional Regions in Them.

Intrinsically disordered regions (IDRs) are protein regions that do not adopt fixed tertiary structures. Since these regions lack ordered three-dimensional structures, they should be excluded from the target portions of homology modeling. IDRs can be predicted from the amino acid sequences, because their amino acid compositions are different from that of the structured domains. This chapter provides a review of the prediction methods of IDRs and a case study of IDR prediction.

Prediction of chaperonin GroE substrates using small structural patterns of proteins.

Molecular chaperones are indispensable proteins that assist the folding of aggregation-prone proteins into their functional native states, thereby maintaining organized cellular systems. Two of the best-characterized chaperones are the Escherichia coli chaperonins GroEL and GroES (GroE), for which in vivo obligate substrates have been identified by proteome-wide experiments. These substrates comprise various proteins but exhibit remarkable structural features. They include a number of α/ß proteins, particularly those adopting the TIM ß/α barrel fold. This observation led us to speculate that GroE obligate substrates share a structural motif. Based on this hypothesis, we exhaustively compared substrate structures with the MICAN alignment tool, which detects common structural patterns while ignoring the connectivity or orientation of secondary structural elements. We selected four (or five) substructures with hydrophobic indices that were mostly included in substrates and excluded in others, and developed a GroE obligate substrate discriminator. The substructures are structurally similar and superimposable on the 2-layer 2α4ß sandwich, the most popular protein substructure, implying that targeting this structural pattern is a useful strategy for GroE to assist numerous proteins. Seventeen false positives predicted by our methods were experimentally examined using GroE-depleted cells, and 9 proteins were confirmed to be novel GroE obligate substrates. Together, these results demonstrate the utility of our common substructure hypothesis and prediction method.

How AlphaFold2 Predicts Conditionally Folding Regions Annotated in an Intrinsically Disordered Protein Database, IDEAL.

AlphaFold2 (AF2) is a protein structure prediction program which provides accurate models. In addition to predicting structural domains, AF2 assigns intrinsically disordered regions (IDRs) by identifying regions with low prediction reliability (pLDDT). Some regions in IDRs undergo disorder-to-order transition upon binding the interaction partner. Here we assessed model structures of AF2 based on the annotations in IDEAL, in which segments with disorder-to-order transition have been collected as Protean Segments (ProSs). We non-redundantly selected ProSs from IDEAL and classified them based on the root mean square deviation to the corresponding region of AF2 models. Statistical analysis identified 11 structural and sequential features, possibly contributing toward the prediction of ProS structures. These features were categorized into two groups: one that contained pLDDT and the other that contained normalized radius of gyration. The typical ProS structures in the former group comprise a long α helix or a whole or part of the structural domain and those in the latter group comprise a short α helix with terminal loops.

Classification of proteins inducing liquid-liquid phase separation: sequential, structural and functional characterization.

Liquid-liquid phase separation (LLPS) within the cell can form biological condensates, which are increasingly recognized to play important roles in various biological processes. Most proteins involved in LLPS are known to be intrinsically disordered proteins containing intrinsically disordered regions (IDRs) with low complexity regions (LCRs). The proteins driving LLPS were selected from databases of LLPS-related proteins and then classified into three classes according to the components in the condensates. Through in silico analyses, we found that proteins in the homo class, those that induce LLPS without partner molecules, have different IDRs and LCRs compared with the reference proteome. In contrast, proteins in the other classes, those that induce LLPS with partner proteins (the hetero class) or nucleic acids (the mixed class), did not show a clear difference to the reference proteome in IDRs and LCRs. The hetero-class proteins contained structural domains to serve protein-protein interactions, and the mixed-class ones had the structural domains associated with nucleic acids. These results suggest that IDRs in the homo-class proteins have unique IDRs, which provide multivalent interaction sites required for LLPS, whereas the hetero- and mixed-class proteins can induce LLPS through the combination of the interaction among LCRs, structural domains and nucleic acids.

Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Evaluating cepharanthine analogues as natural drugs against SARS-CoV-2.

Cepharanthine (CEP) is a natural biscoclaurine alkaloid of plant origin and was recently demonstrated to have anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) activity. In this study, we evaluated whether natural analogues of CEP may act as potential anti-coronavirus disease 2019 drugs. A total of 24 compounds resembling CEP were extracted from the KNApSAcK database, and their binding affinities to target proteins, including the spike protein and main protease of SARS-CoV-2, NPC1 and TPC2 in humans, were predicted via molecular docking simulations. Selected analogues were further evaluated by a cell-based SARS-CoV-2 infection assay. In addition, the efficacies of CEP and its analogue tetrandrine were assessed. A comparison of the docking conformations of these compounds suggested that the diphenyl ester moiety of the molecules was a putative pharmacophore of the CEP analogues.

Current status of structure-based drug repurposing against COVID-19 by targeting SARS-CoV-2 proteins.

More than one and half years have passed, as of August 2021, since the COVID-19 caused by the novel coronavirus named SARS-CoV-2 emerged in 2019. While the recent success of vaccine developments likely reduces the severe cases, there is still a strong requirement of safety and effective therapeutic drugs for overcoming the unprecedented situation. Here we review the recent progress and the status of the drug discovery against COVID-19 with emphasizing a structure-based perspective. Structural data regarding the SARS-CoV-2 proteome has been rapidly accumulated in the Protein Data Bank, and up to 68% of the total amino acid residues encoded in the genome were covered by the structural data. Despite a global effort of in silico and in vitro screenings for drug repurposing, there is only a limited number of drugs had been successfully authorized by drug regulation organizations. Although many approved drugs and natural compounds, which exhibited antiviral activity in vitro, were considered potential drugs against COVID-19, a further multidisciplinary investigation is required for understanding the mechanisms underlying the antiviral effects of the drugs.

Structural Insights into the Regulation of Actin Capping Protein by Twinfilin C-terminal Tail.

Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI.

Crystal structure of human V-1 in the apo form.

V-1, also known as myotrophin, is a 13 kDa ankyrin-repeat protein that binds and inhibits the heterodimeric actin capping protein (CP), which is a key regulator of cytoskeletal actin dynamics. The crystal structure of V-1 in complex with CP revealed that V-1 recognizes CP via residues spanning several ankyrin repeats. Here, the crystal structure of human V-1 is reported in the absence of the specific ligand at 2.3 Šresolution. In the asymmetric unit, the crystal contains two V-1 monomers that exhibit nearly identical structures (Cα r.m.s.d. of 0.47 Å). The overall structures of the two apo V-1 chains are also highly similar to that of CP-bound V-1 (Cα r.m.s.d.s of <0.50 Å), indicating that CP does not induce a large conformational change in V-1. Detailed structural comparisons using the computational program All Atom Motion Tree revealed that CP binding can be accomplished by minor side-chain rearrangements of several residues. These findings are consistent with the known biological role of V-1, in which it globally inhibits CP in the cytoplasm.

Knowledge-based structural models of SARS-CoV-2 proteins and their complexes with potential drugs.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) caused by the novel coronavirus SARS-CoV-2 a pandemic. There is, however, no confirmed anti-COVID-19 therapeutic currently. In order to assist structure-based discovery efforts for repurposing drugs against this disease, we constructed knowledge-based models of SARS-CoV-2 proteins and compared the ligand molecules in the template structures with approved/experimental drugs and components of natural medicines. Our theoretical models suggest several drugs, such as carfilzomib, sinefungin, tecadenoson, and trabodenoson, that could be further investigated for their potential for treating COVID-19.

Protein kinases phosphorylate long disordered regions in intrinsically disordered proteins.

Phosphorylation is a major post-translational modification that plays a central role in signaling pathways. Protein kinases phosphorylate substrates (phosphoproteins) by adding phosphate at Ser/Thr or Tyr residues (phosphosites). A large amount of data identifying and describing phosphosites in phosphoproteins has been reported but the specificity of phosphorylation is not fully resolved. In this report, data of kinase-substrate pairs identified by the Kinase-Interacting Substrate Screening (KISS) method were used to analyze phosphosites in intrinsically disordered regions (IDRs) of intrinsically disordered proteins. We compared phosphorylated and nonphosphorylated IDRs and found that the phosphorylated IDRs were significantly longer than nonphosphorylated IDRs. The phosphorylated IDR is often the longest IDR (71%) in a phosphoprotein when only a single phosphosite exists in the IDR, and when the phosphoprotein has multiple phosphosites in an IDR(s), the phosphosites are primarily localized in a single IDR (78%) and this IDR is usually the longest one (81%). We constructed a stochastic model of phosphorylation to estimate the effect of IDR length. The model that accounted for IDR length produced more realistic results when compared with a model that excluded the IDR length. We propose that the IDR length is a significant determinant for locating kinase phosphorylation sites in phosphoproteins.

All Atom Motion Tree detects side chain-related motions and their coupling with domain motion in proteins.

Structural changes of proteins are closely related with their molecular function. We previously developed a computational tool, Motion Tree (MT), to compare protein structures and describe structural changes using solely the Cα atoms. Here, we have extended MT to incorporate all heavy atoms to analyze side chain-related (SCR) motions. All Atom Motion Tree (AAMT) was applied to 76 proteins that exhibited a simple domain motion identified by MT. AAMT also detected 921 SCR motions. We examined the coupling of domain and SCR motions and classified the structural changes in terms of coupling. The statistical results indicated that it is common for coupled SCR motions to also couple with the domain motion. The classification correlates properties of domain motions and SCR motions. The AAMT results suggest that a large domain motion with a sizable domain boundary is accompanied by SCR motions composed of more than a single residue, which induces further couplings of SCR motions.

Using 1 HN amide temperature coefficients to define intrinsically disordered regions: An alternative NMR method.

This report describes a cost-effective experimental method for determining an intrinsically disordered protein (IDP) region in a given protein sample. In this area, the most popular (and conventional) means is using the amide (1 HN ) NMR signal chemical shift distributed in the range of 7.5-8.5 ppm. For this study, we applied an additional step: analysis of 1 HN chemical shift temperature coefficients (1 HN -CSTCs) of the signals. We measured 1 H-15 N two-dimensional NMR spectra of model IDP samples and ordered samples at four temperatures (288, 293, 298, and 303 K). We derived the 1 HN -CSTC threshold deviation, which gives the best correlation of ordered and disordered regions among the proteins examined (below -3.6 ppb/K). By combining these criteria with the newly optimized chemical shift range (7.8-8.5 ppm), the ratios of both true positive and true negative were improved by approximately 19% (62-81%) compared with the conventional "chemical shift-only" method.

MICAN-SQ: a sequential protein structure alignment program that is applicable to monomers and all types of oligomers.

Motivation: Protein structure alignment is a significant tool to understand evolutionary processes and physicochemical properties of proteins. Important targets of structure alignment are not only monomeric but also oligomeric proteins that sometimes include domain swapping or fusions. Although various protein structural alignment programs have been developed, no method is applicable to any protein pair regardless of the number of chain components and oligomeric states with retaining sequential restrictions: structurally equivalent regions must be aligned in the same order along protein sequences. Results: In this paper, we introduced a new sequential protein structural alignment algorithm MICAN-SQ, which is applicable to protein structures in all oligomeric states. In particular, MICAN-SQ allows the complicated structural alignments of proteins with domain swapping or fusion regions. To validate MICAN-SQ, alignment accuracies were evaluated using curated alignments of monomers and examples of domain swapping, and compared with those of pre-existing protein structural alignment programs. The results of this study show that MICAN-SQ has superior accuracy and robustness in comparison with previous programs and offers limited computational times. We also demonstrate that MICAN-SQ correctly aligns very large complexes and fused proteins. The present computations warrant the consideration of MICAN-SQ for studies of evolutionary and physicochemical properties of monomeric structures and all oligomer types. Availability and implementation: The MICAN program was implemented in C. The source code and executable file can be freely downloaded from http://www.tbp.cse.nagoya-u.ac.jp/MICAN/. Supplementary information: Supplementary data are available at Bioinformatics online.

Discovery of Cryoprotective Activity in Human Genome-Derived Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK2 motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK2 motif serving as a core region for the dehydrins' cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH). All five IDPs that were examined were in the range of 35-45 amino acid residues in length and were equally potent at a concentration of 50 µg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ) domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S-transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA). Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future.

Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding.

A subset of the proteome is prone to aggregate formation, which is prevented by chaperones in the cell. To investigate whether the basic principle underlying the aggregation process is common in prokaryotes and eukaryotes, we conducted a large-scale aggregation analysis of ~500 cytosolic budding yeast proteins using a chaperone-free reconstituted translation system, and compared the obtained data with that of ~3,000 Escherichia coli proteins reported previously. Although the physicochemical properties affecting the aggregation propensity were generally similar in yeast and E. coli proteins, the susceptibility of aggregation in yeast proteins were positively correlated with the presence of intrinsically disordered regions (IDRs). Notably, the aggregation propensity was not significantly changed by a removal of IDRs in model IDR-containing proteins, suggesting that the properties of ordered regions in these proteins are the dominant factors for aggregate formation. We also found that the proteins with longer IDRs were disfavored by E. coli chaperonin GroEL/ES, whereas both bacterial and yeast Hsp70/40 chaperones have a strong aggregation-prevention effect even for proteins possessing IDRs. These results imply that a key determinant to discriminate the eukaryotic proteomes from the prokaryotic proteomes in terms of protein folding would be the attachment of IDRs.

Structural changes of homodimers in the PDB.

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes.

Rules for connectivity of secondary structure elements in protein: Two-layer αß sandwiches.

In protein structures, the fold is described according to the spatial arrangement of secondary structure elements (SSEs: α-helices and ß-strands) and their connectivity. The connectivity or the pattern of links among SSEs is one of the most important factors for understanding the variety of protein folds. In this study, we introduced the connectivity strings that encode the connectivities by using the types, positions, and connections of SSEs, and computationally enumerated all the connectivities of two-layer αß sandwiches. The calculated connectivities were compared with those in natural proteins determined using MICAN, a nonsequential structure comparison method. For 2α-4ß, among 23,000 of all connectivities, only 48 were free from irregular connectivities such as loop crossing. Of these, only 20 were found in natural proteins and the superfamilies were biased toward certain types of connectivities. A similar disproportional distribution was confirmed for most of other spatial arrangements of SSEs in the two-layer αß sandwiches. We found two connectivity rules that explain the bias well: the abundances of interlayer connecting loops that bridge SSEs in the distinct layers; and nonlocal ß-strand pairs, two spatially adjacent ß-strands located at discontinuous positions in the amino acid sequence. A two-dimensional plot of these two properties indicated that the two connectivity rules are not independent, which may be interpreted as a rule for the cooperativity of proteins.