Exploration of novel αß-protein folds through de novo design.

A fundamental question in protein evolution is whether nature has exhaustively sampled nearly all possible protein folds throughout evolution, or whether a large fraction of the possible folds remains unexplored. To address this question, we defined a set of rules for ß-sheet topology to predict novel αß-folds and carried out a systematic de novo protein design exploration of the novel αß-folds predicted by the rules. The designs for all eight of the predicted novel αß-folds with a four-stranded ß-sheet, including a knot-forming one, folded into structures close to the design models. Further, the rules predicted more than 10,000 novel αß-folds with five- to eight-stranded ß-sheets; this number far exceeds the number of αß-folds observed in nature so far. This result suggests that a vast number of αß-folds are possible, but have not emerged or have become extinct due to evolutionary bias.

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.

Enumeration and comprehensive in-silico modeling of three-helix bundle structures composed of typical αα-hairpins.

BACKGROUND: The design of protein structures from scratch requires special attention to the combination of the types and lengths of the secondary structures and the loops required to build highly designable backbone structure models. However, it is difficult to predict the combinations that result in globular and protein-like conformations without simulations. In this study, we used single-chain three-helix bundles as simple models of protein tertiary structures and sought to thoroughly investigate the conditions required to construct them, starting from the identification of the typical αα-hairpin motifs. RESULTS: First, by statistical analysis of naturally occurring protein structures, we identified three αα-hairpins motifs that were specifically related to the left- and right-handedness of helix-helix packing. Second, specifying these αα-hairpins motifs as junctions, we performed sequence-independent backbone-building simulations to comparatively build single-chain three-helix bundle structures and identified the promising combinations of the length of the α-helix and αα-hairpins types that results in tight packing between the first and third α-helices. Third, using those single-chain three-helix bundle backbone structures as template structures, we designed amino acid sequences that were predicted to fold into the target topologies, which supports that the compact single-chain three-helix bundles structures that we sampled show sufficient quality to allow amino-acid sequence design. CONCLUSION: The enumeration of the dominant subsets of possible backbone structures for small single-chain three-helical bundle topologies revealed that the compact foldable structures are discontinuously and sparsely distributed in the conformational space. Additionally, although the designs have not been experimentally validated in the present research, the comprehensive set of computational structural models generated also offers protein designers the opportunity to skip building similar structures by themselves and enables them to quickly focus on building specialized designs using the prebuilt structure models. The backbone and best design models in this study are publicly accessible from the following URL: https://doi.org/10.5281/zenodo.4321632 .

A prospective compound screening contest identified broader inhibitors for Sirtuin 1.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Characteristics of interactions at protein segments without non-local intramolecular contacts in the Protein Data Bank.

The principle of three-dimensional protein structure formation is a long-standing conundrum in structural biology. A globular domain of a soluble protein is formed by a network of atomic contacts among amino acid residues, but regions without intramolecular non-local contacts are often observed in the protein structure, especially in loop, linker, and peripheral segments with secondary structures. Although these regions can play key roles for protein function as interfaces for intermolecular interactions, their nature remains unclear. Here, we termed protein segments without non-local contacts as floating segments and sought them in tens of thousands of entries in the Protein Data Bank. As a result, we found that 0.72% of residues are in floating segments. Regarding secondary structural elements, coil structures are enriched in floating segments, especially for long segments. Interactions with polypeptides and polynucleotides, but not chemical compounds, are enriched in floating segments. The amino acid preferences of floating segments are similar to those of surface residues, with exceptions; the small side chain amino acids, Gly and Ala, are preferred, and some charged side chains, Arg and His, are disfavored for floating segments compared to surface residues. Our comprehensive characterization of floating segments may provide insights into understanding protein sequence-structure-function relationships.

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.

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.

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.

An iterative compound screening contest method for identifying target protein inhibitors using the tyrosine-protein kinase Yes.

We propose a new iterative screening contest method to identify target protein inhibitors. After conducting a compound screening contest in 2014, we report results acquired from a contest held in 2015 in this study. Our aims were to identify target enzyme inhibitors and to benchmark a variety of computer-aided drug discovery methods under identical experimental conditions. In both contests, we employed the tyrosine-protein kinase Yes as an example target protein. Participating groups virtually screened possible inhibitors from a library containing 2.4 million compounds. Compounds were ranked based on functional scores obtained using their respective methods, and the top 181 compounds from each group were selected. Our results from the 2015 contest show an improved hit rate when compared to results from the 2014 contest. In addition, we have successfully identified a statistically-warranted method for identifying target inhibitors. Quantitative analysis of the most successful method gave additional insights into important characteristics of the method used.

Importance of consensus region of multiple-ligand templates in a virtual screening method.

We discuss methods and ideas of virtual screening (VS) for drug discovery by examining the performance of VS-APPLE, a recently developed VS method, which extensively utilizes the tendency of single binding pockets to bind diversely different ligands, i.e. promiscuity of binding pockets. In VS-APPLE, multiple ligands bound to a pocket are spatially arranged by maximizing structural overlap of the protein while keeping their relative position and orientation with respect to the pocket surface, which are then combined into a multiple-ligand template for screening test compounds. To greatly reduce the computational cost, comparison of test compound structures are made only with limited regions of the multiple-ligand template. Even when we use the narrow regions with most densely populated atoms for the comparison, VSAPPLE outperforms other conventional VS methods in terms of Area Under the Curve (AUC) measure. This region with densely populated atoms corresponds to the consensus region among multiple ligands. It is typically observed that expansion of the sampled region including more atoms improves screening efficiency. However, for some target proteins, considering only a small consensus region is enough for the effective screening of test compounds. These results suggest that the performance test of VS methods sheds light on the mechanisms of protein-ligand interactions, and elucidation of the protein-ligand interactions should further help improvement of VS methods.

Synthesis of multiply substituted 1,6-dihydropyridines through Cu(I)-catalyzed 6-endo cyclization.

Copper-catalyzed 6-endo cyclization of N-propargylic ß-enaminocarbonyls was developed for the synthesis of oxidation-labile 1,6-dihydropyridines. This synthetic method allows flexible and regio-defined assembly of various substituents at the N1, C2, C3, C4, and C6 positions of 1,6-dihydropyridines under mild conditions.

How a spatial arrangement of secondary structure elements is dispersed in the universe of protein folds.

It has been known that topologically different proteins of the same class sometimes share the same spatial arrangement of secondary structure elements (SSEs). However, the frequency by which topologically different structures share the same spatial arrangement of SSEs is unclear. It is important to estimate this frequency because it provides both a deeper understanding of the geometry of protein folds and a valuable suggestion for predicting protein structures with novel folds. Here we clarified the frequency with which protein folds share the same SSE packing arrangement with other folds, the types of spatial arrangement of SSEs that are frequently observed across different folds, and the diversity of protein folds that share the same spatial arrangement of SSEs with a given fold, using a protein structure alignment program MICAN, which we have been developing. By performing comprehensive structural comparison of SCOP fold representatives, we found that approximately 80% of protein folds share the same spatial arrangement of SSEs with other folds. We also observed that many protein pairs that share the same spatial arrangement of SSEs belong to the different classes, often with an opposing N- to C-terminal direction of the polypeptide chain. The most frequently observed spatial arrangement of SSEs was the 2-layer α/ß packing arrangement and it was dispersed among as many as 27% of SCOP fold representatives. These results suggest that the same spatial arrangements of SSEs are adopted by a wide variety of different folds and that the spatial arrangement of SSEs is highly robust against the N- to C-terminal direction of the polypeptide chain.

MICAN: a protein structure alignment algorithm that can handle Multiple-chains, Inverse alignments, C(α) only models, Alternative alignments, and Non-sequential alignments.

BACKGROUND: Protein pairs that have the same secondary structure packing arrangement but have different topologies have attracted much attention in terms of both evolution and physical chemistry of protein structures. Further investigation of such protein relationships would give us a hint as to how proteins can change their fold in the course of evolution, as well as a insight into physico-chemical properties of secondary structure packing. For this purpose, highly accurate sequence order independent structure comparison methods are needed. RESULTS: We have developed a novel protein structure alignment algorithm, MICAN (a structure alignment algorithm that can handle Multiple-chain complexes, Inverse direction of secondary structures, Cα only models, Alternative alignments, and Non-sequential alignments). The algorithm was designed so as to identify the best structural alignment between protein pairs by disregarding the connectivity between secondary structure elements (SSE). One of the key feature of the algorithm is utilizing the multiple vector representation for each SSE, which enables us to correctly treat bent or twisted nature of long SSE. We compared MICAN with other 9 publicly available structure alignment programs, using both reference-dependent and reference-independent evaluation methods on a variety of benchmark test sets which include both sequential and non-sequential alignments. We show that MICAN outperforms the other existing methods for reproducing reference alignments of non-sequential test sets. Further, although MICAN does not specialize in sequential structure alignment, it showed the top level performance on the sequential test sets. We also show that MICAN program is the fastest non-sequential structure alignment program among all the programs we examined here. CONCLUSIONS: MICAN is the fastest and the most accurate program among non-sequential alignment programs we examined here. These results suggest that MICAN is a highly effective tool for automatically detecting non-trivial structural relationships of proteins, such as circular permutations and segment-swapping, many of which have been identified manually by human experts so far. The source code of MICAN is freely download-able at http://www.tbp.cse.nagoya-u.ac.jp/MICAN.

Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674-683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.