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N-Linked glycosylation is crucial for various biological processes such as protein folding, immune response, and cellular transport. Traditional experimental methods for determining N-linked glycosylation sites entail substantial time and labor investment, which has led to the development of computational approaches as a more efficient alternative. However, due to the limited availability of 3D structural data, existing prediction methods often struggle to fully utilize structural information and fall short in integrating sequence and structural information effectively. Motivated by the progress of protein pretrained language models (pLMs) and the breakthrough in protein structure prediction, we introduced a high-accuracy model called CoNglyPred. Having compared various pLMs, we opt for the large-scale pLM ESM-2 to extract sequence embeddings, thus mitigating certain limitations associated with manual feature extraction. Meanwhile, our approach employs a graph transformer network to process the 3D protein structures predicted by AlphaFold2. The final graph output and ESM-2 embedding are intricately integrated through a co-attention mechanism. Among a series of comprehensive experiments on the independent test dataset, CoNglyPred outperforms state-of-the-art models and demonstrates exceptional performance in case study. In addition, we are the first to report the uncertainty of N-linked glycosylation predictors using expected calibration error and expected uncertainty calibration error., (© 2024 Wiley‐VCH GmbH.)

Background: ​​The genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies., Description: In response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework., Conclusion: FPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering., (© 2024 Wiley‐VCH GmbH.)

Sialic acids play crucial roles in cell surface glycans of both eukaryotic and prokaryotic organisms, mediating various biological processes, including cell-cell interactions, development, immune response, oncogenesis and host-pathogen interactions. This review focuses on the β-anomeric form of N-acetylneuraminic acid (Neu5Ac), particularly its binding affinity towards various proteins, as elucidated by solved protein structures. Specifically, we delve into the binding mechanisms of Neu5Ac to proteins involved in sequestering and transporting Neu5Ac in Gram-negative bacteria, with implications for drug design targeting these proteins as antimicrobial agents. Unlike the initial assumptions, structural analyses revealed significant variability in the Neu5Ac binding pockets among proteins, indicating diverse evolutionary origins and binding modes. By comparing these findings with existing structures from other systems, we can effectively highlight the intricate relationship between protein structure and Neu5Ac recognition, emphasizing the need for tailored drug design strategies to inhibit Neu5Ac-binding proteins across bacterial species., (open access.)

MltG, a membrane-bound lytic transglycosylase, has roles in terminating glycan polymerization in peptidoglycan and incorporating glycan chains into the cell wall, making it significant in bacterial cell-wall biosynthesis and remodeling. This study provides the first reported MltG structure from Mycobacterium abscessus (maMltG), a superbug that has high antibiotic resistance. Our structural and biochemical analyses revealed that MltG has a flexible peptidoglycan-binding domain and exists as a monomer in solution. Further, the putative active site of maMltG was disclosed using structural analysis and sequence comparison. Overall, this study contributes to our understanding of the transglycosylation reaction of the MltG family, aiding the design of next-generation antibiotics targeting M. abscessus., (open access.)

The analysis of pressure induced changes in the chemical shift of proteins allows statements on structural fluctuations proteins exhibit at ambient pressure. The inherent issue of separating general pressure effects from structural related effects on the pressure dependence of chemical shifts has so far been addressed by considering the characteristics of random coil peptides on increasing pressure. In this work, chemically and pressure denatured states of the cold shock protein B from Bacillus subtilis (BsCspB) have been assigned in 2D 1 H- 15 N HSQC NMR spectra and their dependence on increasing hydrostatic pressure has been evaluated. The pressure denatured polypeptide chain has been used to separate general from structural related effects on 1 H and 15 N chemical shifts of native BsCspB and the implications on the interpretation of pressure induced changes in the chemical shift regarding the structure of BsCspB are discussed. It has been found that the ensemble of unstructured conformations of BsCspB shows different responses to increasing pressure than random coil peptides do. Thus, the approach used for considering the general effects that arise when hydrostatic pressure increases changes the structural conclusions that are drawn from high pressure NMR spectroscopic experiments that rely on the analysis of chemical shifts., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Ultra-intense, ultra-fast X-ray free-electron lasers (XFELs) enable the imaging of single protein molecules under ambient temperature and pressure. A crucial aspect of structure reconstruction involves determining the relative orientations of each diffraction pattern and recovering the missing phase information. In this paper, we introduce a predicted model-aided algorithm for orientation determination and phase retrieval, which has been tested on various simulated datasets and has shown significant improvements in the success rate, accuracy and efficiency of XFEL data reconstruction., (open access.)

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg 2+ ), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research., (© 2024 Wiley-VCH GmbH.)

Oryza sativa L. is the world's most essential and economically important food crop. Climate change and ecological imbalances make rice plants vulnerable to abiotic and biotic stresses, threatening global food security. The Alfin-like (AL) transcription factor family plays a crucial role in plant development and stress responses. This study comprehensively analyzed this gene family and their expression profiles in rice, revealing nine AL genes, classifying them into three distinct groups based on phylogenetic analysis and identifying four segmental duplication events. RNA-seq data analysis revealed high expression levels of OsALs in different tissues, growth stages, and their responsiveness to stresses. RT-qPCR data showed significant expression of OsALs in different abiotic stresses. Identification of potential cis -regulatory elements in promoter regions has also unveiled their involvement. Tertiary structures of the proteins were predicted. These findings would lay the groundwork for future research to reveal their molecular mechanism in stress tolerance and plant development., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB-TcC subcomplex that makes a hollow shell. This TcB-TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB-TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization., (open access.)

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca 2+ which agrees with acidic residues found to coordinate Ca 2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos., (open access.)

Problems with minced pork include water release and low gel strength. This study aimed to investigate the effect of treatments with κ-carrageenan (κ-CAR), egg white powder (EWP), wheat gluten (WG), soy isolate protein (SPI), and a combination of these treatments on the gel properties and protein structures of minced pork. The cooking loss and trapped water within minced pork increased when additives were incorporated; in particular, the SPI group reached 1.31 ± 0.01% and 91.42 ± 0.20%. The hardness and chewiness of minced pork reached their maximum values (38.91 ± 0.80 N, 14.73 ± 0.41 N) when the WG was added. The κ-CAR/WG-minced pork gel network structure was the densest and most stable, characterized by increased hydrophobic interactions, disulfide bonds in the mince gel, and enthalpy value. The α-helix content with κ-CAR/WG treatment decreased from 27% to 7.8%, transforming into other secondary structures. This suggests that the addition of κ-CAR/WG can be a more effective combination for improving the quality of minced pork.

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross-reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ-1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'-alkyl,N-cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with effective target engagement in cells. Notably, transitioning from 5-membered pyrrolidines to 6-membered heterocycles eliminated PARK7 binding and introduced context-dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound-induced conformational restriction of the cross-over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria., (open access.)

Highly accurate protein structure prediction can generate accurate models of protein and protein-protein complexes in X-ray crystallography. However, the question of how to make more effective use of predicted models for completing structure analysis, and which strategies should be employed for the more challenging cases such as multi-helical structures, multimeric structures and extremely large structures, both in the model preparation and in the completion steps, remains open for discussion. In this paper, a new strategy is proposed based on the framework of direct methods and dual-space iteration, which can greatly simplify the pre-processing steps of predicted models both in normal and in challenging cases. Following this strategy, full-length models or the conservative structural domains could be used directly as the starting model, and the phase error and the model bias between the starting model and the real structure would be modified in the direct-methods-based dual-space iteration. Many challenging cases (from CASP14) have been tested for the general applicability of this constructive strategy, and almost complete models have been generated with reasonable statistics. The hybrid strategy therefore provides a meaningful scheme for X-ray structure determination using a predicted model as the starting point., (open access.)

We present a de novo transcriptome assembly for the non-model Antarctic polychaete worm Microspio moorei (Spionidae) collected during Antarctic field expedition in Fildes Bay, King George Island, Antarctic Peninsula, in 2017. Here, we report the first transcriptome reference array for Microspio spp. The gene sequences of the spionid worm were annotated from a wide range of functions (i.e., biological, and metabolic processes, catalytic processes, and catalytic activity). HSP70, HSP90 SOD and CAT families were compared to reported annelid transcriptomes and proteomes. The phylogenetic analysis using COI, 16S, and 18S markers effectively clusters the species within the family. However, it also casts uncertainty on the monophyletic nature of the Microspio genera, indicating the necessity for additional data and potentially requiring a reevaluation of its grouping. Within these protein families, 3D model software was used to create one representative of their protein structures. Structural predictions were compared with related reported annelids living at different temperatures and a human X-ray reference. We found structural differences (RMSE >1.8) between the human HSP proteins but no significant differences between the polychaete-predicted proteins (RMSE <1.2). These results encourage further research of heat stress-related proteins, the development of genetic markers for climate change-induced temperature stress, and the study of the underlying mechanisms of the heat response. Moreover, these results motivate the extension of these findings to congeneric species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Javier Alvarez reports financial support was provided by EAFIT University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ice-binding proteins (IBPs) are proteins that have the remarkable ability to bind to ice, and their study has intrigued researchers for decades. This chapter explores the importance of structural biology in understanding IBPs and highlights the significant contributions of IBPs to the field of structural biology. The structures of various IBPs from different organisms have been elucidated, revealing key elements involved in ice binding. Structural biology techniques, including nuclear magnetic resonance (NMR) spectroscopy, transmission electron cryo-microscopy (cryo-EM), and X-ray crystallography, play crucial roles in solving protein structures. This article focuses on X-ray crystallography as a tool for investigating IBP structures, providing insights into its theoretical and practical aspects, experimental workflows, and common pitfalls to avoid., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Regio- and chemoselective C-H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C-H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes., (© 2023 Wiley-VCH GmbH.)

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP 5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP 5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The rapid evolution of protein structure prediction tools has significantly broadened access to protein structural data. Although predicted structure models have the potential to accelerate and impact fundamental and translational research significantly, it is essential to note that they are not validated and cannot be considered the ground truth. Thus, challenges persist, particularly in capturing protein dynamics, predicting multi-chain structures, interpreting protein function, and assessing model quality. Interdisciplinary collaborations are crucial to overcoming these obstacles. Databases like the AlphaFold Protein Structure Database, the ESM Metagenomic Atlas, and initiatives like the 3D-Beacons Network provide FAIR access to these data, enabling their interpretation and application across a broader scientific community. Whilst substantial advancements have been made in protein structure prediction, further progress is required to address the remaining challenges. Developing training materials, nurturing collaborations, and ensuring open data sharing will be paramount in this pursuit. The continued evolution of these tools and methodologies will deepen our understanding of protein function and accelerate disease pathogenesis and drug development discoveries., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

The N2A segment of titin functions as a pivotal hub for signal transduction and interacts with various proteins involved in structural support, chaperone activities, and transcriptional regulation. Notably, the "unique N2A" (UN2A) subdomain has been shown to interact with the stress-regulated cardiac ankyrin repeat protein (CARP), which contributes to the regulation of sarcomeric stiffness. Previously, the UN2A domain's three-dimensional structure was modelled based on its secondary structure content identified by NMR spectroscopy, considering the domain in isolation. In this study, we report experimental long-range distance distributions by electron paramagnetic resonance (EPR) spectroscopy between the three helixes within the UN2A domain linked to the immunoglobulin domain I81 in the presence and absence of CARP. The data confirm the central three-helix bundle fold of UN2A and show that this adopts a compact and stable conformation in absence of CARP. After binding to CARP, no significant conformational change was observed, suggesting that the UN2A domain retains its structure upon binding to CARP thereby, mediating the interaction approximately as a rigid-body., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Magnesium transporters (MGTs) regulate magnesium absorption, transport, and redistribution in higher plants. To investigate the role of the Oryza sativa MGTs gene family members under salt stress, this study analyzed the protein properties, gene structure, phylogenetic relationship, synteny patterns, expression, and co-expression networks of 23 non-redundant OsMGT . The evolutionary relationship of the OsMGT gene family was fully consistent with their functional domain, and were divided into three main classes based on the conserved domain: MMgT, CorA-like, and NIPA. The α/β patterns in the protein structures were highly similar in the CorA-like and NIPA members, with the conserved structures in the Mg 2+ -binding and catalytic regions. The CorA-like clade-related proteins demonstrated the highest numbers of protein channels with Pro, Ser, Lys, Gly, and Tyr, as the critical binding residues. The expression analysis of OsMGT genes in various tissues showed that MGTs ' gene family may possess critical functions during rice development. Gene expression analysis of candidate OsMGT using reverse-transcription quantitative real-time PCR (RT-qPCR) found that four OsMGT genes exhibited different expression patterns in salt-sensitive and salt-tolerant rice genotypes. We hypothesize that the OsMGT gene family members may be involved in responses to salt stress. These findings could be useful for further functional investigation of MGTs as well as defining their involvement in abiotic stress studies., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03735-4., Competing Interests: Conflict of interestThe authors declare no conflict of interest, financial, or otherwise., (© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells., (© 2023 Wiley-VCH GmbH.)

Plasmodium vivax malaria affects 14 million people each year. Its invasion requires interactions between the parasitic Duffy-binding protein ( Pv DBP) and the N-terminal extracellular domain (ECD1) of the host's Duffy antigen/receptor for chemokines (DARC). ECD1 is highly flexible and intrinsically disordered, therefore it can adopt different conformations. We computationally modeled the challenging ECD1 local structure. With T-REMD simulations, we sampled its dynamic behavior and collected its most representative conformations. Our results suggest that most of the DARC ECD1 domain remains in a disordered state during the simulated time. Globular local conformations are found in the analyzed local free-energy minima. These globular conformations share an α-helix spanning residues Ser18 to Ser29 and in many cases they comprise an antiparallel β-sheet, whose β-strands are formed around residues Leu10 and Ala49. The formation of a parallel β-sheet is almost negligible. So far, progress in understanding the mechanisms forming the basis of the P. vivax malaria infection of reticulocytes has been hampered by experimental difficulties, along with a lack of DARC structural information. Our collection of the most probable ECD1 structural conformations will help to advance modeling of the DARC structure and to explore DARC-ECD1 interactions with a range of physiological and pathological ligands.

Many proteins exert their function by switching among different structures. Knowing the conformational ensembles affiliated with these states is critical to elucidate key mechanistic aspects that govern protein function. While experimental determination efforts are still bottlenecked by cost, time, and technical challenges, the machine-learning technology AlphaFold showed near experimental accuracy in predicting the three-dimensional structure of monomeric proteins. However, an AlphaFold ensemble of models usually represents a single conformational state with minimal structural heterogeneity. Consequently, several pipelines have been proposed to either expand the structural breadth of an ensemble or bias the prediction toward a desired conformational state. Here, we analyze how those pipelines work, what they can and cannot predict, and future directions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The scoring models used for protein structure modeling and ranking are mainly divided into unified field and protein-specific scoring functions. Although protein structure prediction has made tremendous progress since CASP14, the modeling accuracy still cannot meet the requirements to a certain extent. Especially, accurate modeling of multi-domain and orphan proteins remains a challenge. Therefore, an accurate and efficient protein scoring model should be developed urgently to guide the protein structure folding or ranking through deep learning. In this work, we propose a protein structure global scoring model based on equivariant graph neural network (EGNN), named GraphGPSM, to guide protein structure modeling and ranking. We construct an EGNN architecture, and a message passing mechanism is designed to update and transmit information between nodes and edges of the graph. Finally, the global score of the protein model is output through a multilayer perceptron. Residue-level ultrafast shape recognition is used to describe the relationship between residues and the overall structure topology, and distance and direction encoded by Gaussian radial basis functions are designed to represent the overall topology of the protein backbone. These two features are combined with Rosetta energy terms, backbone dihedral angles and inter-residue distance and orientations to represent the protein model and embedded into the nodes and edges of the graph neural network. The experimental results on the CASP13, CASP14 and CAMEO test sets show that the scores of our developed GraphGPSM have a strong correlation with the TM-score of the models, which are significantly better than those of the unified field score function REF2015 and the state-of-the-art local lDDT-based scoring models ModFOLD8, ProQ3D and DeepAccNet, etc. The modeling experimental results on 484 test proteins demonstrate that GraphGPSM can greatly improve the modeling accuracy. GraphGPSM is further used to model 35 orphan proteins and 57 multi-domain proteins. The results show that the average TM-score of the models predicted by GraphGPSM is 13.2 and 7.1% higher than that of the models predicted by AlphaFold2. GraphGPSM also participates in CASP15 and achieves competitive performance in global accuracy estimation., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Metal-binding proteins are essential for the vital activities and engage in their roles by acting in concert with metal cations. MbPA (The Metal-binding Protein Atlas) is the most comprehensive resource up to now dedicated to curating metal-binding proteins. Currently, it contains 106,373 entries and 440,187 sites related to 54 metals and 8169 species. Users can view all metal-binding proteins and species-specific proteins in MbPA. There are also metal-proteomics data that quantitatively describes protein expression in different tissues and organs. By analyzing the data of the amino acid residues at the metal-binding site, it is found that about 80% of the metal ions tend to bind to cysteine, aspartic acid, glutamic acid, and histidine. Moreover, we use Diversity Measure to confirm that the diversity of metal-binding is specific in different area of periodic table, and further elucidate the binding modes of 19 transition metals on 20 amino acids. In addition, MbPA also embraces 6855 potential pathogenic mutations related to metalloprotein. The resource is freely available at http://bioinfor.imu.edu.cn/mbpa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complexes. However, the progress of in vivo CXMS studies has been limited by cross-linking biocompatibility and data analysis. Herein, a glycosidic bond-based MS-cleavable cross-linker of trehalose disuccinimidyl ester (TDS) was designed and synthesized, which was fragmented in MS under CID/HCD to simplify the cross-linked peptides into conventional single peptides via selective cleavage between glycosidic and peptide bonds under individual MS collision energy. Consequently, the cross-linking identification accuracy and throughput were significantly enhanced, and the popular MS mode of stepped HCD was allowed. In addition, TDS showed proper cell-penetrating properties while being highly water-soluble, making it non-DMSO dependent during solubilization. Collectively, TDS provides a promising toolkit for CXMS characterization of living systems with high biocompatibility and accuracy., (© 2023 Wiley-VCH GmbH.)

Prevalent in nature, protein oligomers play critical roles both physiologically and pathologically. The multimeric nature and conformational transiency of protein oligomers greatly complicate a more detailed glimpse into the molecular structure as well as function. In this minireview, the oligomers are classified and described on the basis of biological function, toxicity, and application. We also define the bottlenecks in recent oligomer studies and further review numerous frontier methods for engineering protein oligomers. Progress is being made on many fronts for a wide variety of applications, and protein grafting is highlighted as a promising and robust method for oligomer engineering. These advances collectively allow the engineering and design of stabilized oligomers that bring us one step closer to understanding their biological functions, toxicity, and a wide range of applications., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether, and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

This paper presents a novel framework, called PSAC-PDB, for analyzing and classifying protein structures from the Protein Data Bank (PDB). PSAC-PDB first finds, analyze and identifies protein structures in PDB that are similar to a protein structure of interest using a protein structure comparison tool. Second, the amino acids (AA) sequences of identified protein structures (obtained from PDB), their aligned amino acids (AAA) and aligned secondary structure elements (ASSE) (obtained by structural alignment), and frequent AA (FAA) patterns (discovered by sequential pattern mining), are used for the reliable detection/classification of protein structures. Eleven classifiers are used and their performance is compared using six evaluation metrics. Results show that three classifiers perform well on overall, and that FAA patterns can be used to efficiently classify protein structures in place of providing the whole AA sequences, AAA or ASSE. Furthermore, better classification results are obtained using AAA of protein structures rather than AA sequences. PSAC-PDB also performed better than state-of-the-art approaches for SARS-CoV-2 genome sequences classification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Eukaryotic transcription factors (TFs) are the final integrators of a complex molecular feedback mechanism that interfaces with the genome, consolidating information for transcriptional regulation. TFs consist of both structured DNA-binding domains and long intrinsically disordered regions (IDRs) embedded with motifs linked to transcriptional control. It is now well established that the dynamic multifunctionality of IDRs is the basis for a wide spectrum of TF functions necessary to navigate and regulate the human genome. This review dissects the chemical features of TF IDRs that endow them with structural plasticity that is central to their functions in the nucleus. Sequence analysis of a set of over 1600 human TFs through AlphaFold was used to identify key features of their IDRs. Recent studies were then highlighted to illustrate IDR involvement in processes such as protein interactions, DNA binding and specificity, chromatin opening, and phase separation. To expand our understanding of TF functions, future directions are suggested for integrating experiments and simulations, from in vitro to living systems., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Germination significantly increased the nutrient composition, total phenolic content, and antioxidant activity of the chickpea flour cultivars studied (GNG-469 and GNG-1581). Protein and starch digestibilities were significantly improved in germinated chickpea flour. Germinated chickpea flour formed gels that had a shear modulus about 60% that of the non-germinated ones. X-ray diffraction analysis indicated that both A and B type polymorphs were present in the chickpea flour cultivars. Germination significantly reduced the relative crystallinity of the chickpea flour cultivars, from around 33 to 27% for GNG 469 and around 30 to 25% for GNG 1581. Protein secondary structures showed increase in β-sheets and random coils content in chickpea cultivars during germination. Phenolic acid profiling showed a decrease in the concentration of ellagic, p-coumaric, p-hydroxybenzoic, and caffeic acids but an increase in gallic, p-coumaric, and ferulic acids after germination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

It is essential to understand the mechanism of action of ultrasound synergistic free radical oxidation to promote covalent reactions between proteins and polyphenols. (-)-epigallo-catechin 3-gallate (EGCG) with rich bioactivity could be used to increase the functional properties of cereal protein-gliadin (GL). This study systematically explored the role of ultrasound treatment (US) on the binding mechanisms of GL and EGCG. Electrophoresis and high-performance liquid chromatography (HPLC) confirmed the greater molecular mass of the covalent complexes in the ultrasound environment. Quantitative analysis by the phenol content revealed that the ultrasound environment increased the EGCG content in the covalent complex by 15.08 mg/g of protein. The changes in the spatial structure of the proteins were indicated by Fourier infrared and ultraviolet spectroscopy. Additionally, scanning electron microscopy (SEM) and atomic force microscopy (AFM) found that US disrupted the aggregation of GL and the clustered structure of the covalent complexes. The results demonstrated that the water solubility of ultrasonic conjugates was significantly increased by 8.8-64.19%, the digestion rate was more efficient, and the radical scavenging capacity was twice that of GL. This research contributes to the theoretical basis for broadening the application of polyphenols in modifying protein.

We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins., (© 2023 Wiley-VCH GmbH.)

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH., (open access.)

Bacterial biofilms are notorious for their ability to protect bacteria from environmental challenges, most importantly the action of antibiotics. Bacillus subtilis is an extensively studied model organism used to understand the process of biofilm formation. A complex network of principal regulatory proteins including Spo0A, AbrB, AbbA, Abh, SinR, SinI, SlrR, and RemA, work in concert to transition B. subtilis from the free-swimming planktonic state to the biofilm state. In this review, we explore, connect, and summarize decades worth of structural and biochemical studies that have elucidated this protein signaling network. Since structure dictates function, unraveling aspects of protein molecular mechanisms will allow us to devise ways to exploit critical features of the biofilm regulatory pathway, such as possible therapeutic intervention. This review pools our current knowledge base of B. subtilis biofilm regulatory proteins and highlights potential therapeutic intervention points., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Compared to the disulfide bond, other naturally occurring intramolecular crosslinks have received little attention, presumably due to their rarity in the vast protein space. Here we presented examples of natural non-disulfide crosslinks, which we refer to as orthogonal crosslinks, emphasizing their effect on protein topology and function. We summarize recent efforts on expanding orthogonal crosslinks by using either the enzymes that catalyze protein circularization or the genetic code expansion strategy to add electrophilic amino acids site-specifically in proteins. The advantages and disadvantages of each method are discussed, along with their applications to generate novel protein topology and function. In particular, we highlight our recent work on spontaneous orthogonal crosslinking, in which a carbamate-based crosslink was generated in situ, and its applications in designing orthogonally crosslinked domain antibodies with their topology-mimicking bacterial adhesins., (© 2022 Wiley-VCH GmbH.)

Immunogenicity is an important concern to therapeutic antibodies during antibody design and development. Based on the co-crystal structures of idiotypic antibodies and their antibodies, one can see that anti-idiotypic antibodies usually bind the complementarity-determining regions (CDR) of idiotypic antibodies. Sequence and structural features, such as cavity volume at the CDR region and hydrophobicity of CDR-H3 loop region, were identified for distinguishing immunogenic antibodies from non-immunogenic antibodies. These features were integrated together with a machine learning platform to predict immunogenicity for humanized and fully human therapeutic antibodies (PITHA). This method achieved an accuracy of 83% in a leave-one-out experiment for 29 therapeutic antibodies with available crystal structures. The web server of this method is accessible at http://mabmedicine.com/PITHA or http://sysbio.unl.edu/PITHA . This method, as a step of computer-aided antibody design, helps evaluate the safety of new therapeutic antibody, which can save time and money during the therapeutic antibody development., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Heavy Chain Only Antibodies are specific to Camelid species. Despite the lack of the light chain variable domain, their heavy chain variable domain (VH) domain, named V H H or nanobody, has promising potential applications in research and therapeutic fields. The structural study of V H H is therefore of great interest. Unfortunately, considering the huge amount of sequences that might be produced, only about one thousand of V H H experimental structures are publicly available in the Protein Data Bank, implying that structural model prediction of V H H is a necessary alternative to obtaining 3D information besides its sequence. The present study aims to assess and compare the quality of predictions from different modelling methodologies. Established comparative & homology modelling approaches to recent Deep Learning-based modelling strategies were applied, i.e. Modeller using single or multiple structural templates, ModWeb, SwissModel (with two evaluation schema), RoseTTAfold, AlphaFold 2 and NanoNet. The prediction accuracy was evaluated using RMSD, TM-score, GDT-TS, GDT-HA and Protein Blocks distance metrics. Besides the global structure assessment, we performed specific analyses of Frameworks and CDRs structures. We observed that AlphaFold 2 and especially NanoNet performed better than the other evaluated softwares. Importantly, we performed molecular dynamics simulations of an experimental structure and a NanoNet predicted model of a V H H in order to compare the global structural flexibility and local conformations using Protein Blocks. Despite rather similar structures, substantial differences in dynamical properties were observed, which underlies the complexity of the task of model evaluation.Communicated by Ramaswamy H. Sarma.

Recent breakthroughs in protein structure prediction demarcate the start of a new era in structural bioinformatics. Combined with various advances in experimental structure determination and the uninterrupted pace at which new structures are published, this promises an age in which protein structure information is as prevalent and ubiquitous as sequence. Machine learning in protein bioinformatics has been dominated by sequence-based methods, but this is now changing to make use of the deluge of rich structural information as input. Machine learning methods making use of structures are scattered across literature and cover a number of different applications and scopes; while some try to address questions and tasks within a single protein family, others aim to capture characteristics across all available proteins. In this review, we look at the variety of structure-based machine learning approaches, how structures can be used as input, and typical applications of these approaches in protein biology. We also discuss current challenges and opportunities in this all-important and increasingly popular field., Competing Interests: We have no conflicts of interest to disclose., (© 2023 The Authors.)

Proline residues within proteins lack a traditional hydrogen bond donor. However, the hydrogens of the proline ring are all sterically accessible, with polarized C-H bonds at Hα and Hδ that exhibit greater partial positive character and can be utilized as alternative sites for molecular recognition. C-H/O interactions, between proline C-H bonds and oxygen lone pairs, have been previously identified as modes of recognition within protein structures and for higher-order assembly of protein structures. In order to better understand intermolecular recognition of proline residues, a series of proline derivatives was synthesized, including 4R-hydroxyproline nitrobenzoate methyl ester, acylated on the proline nitrogen with bromoacetyl and glycolyl groups, and Boc-4S-(4-iodophenyl)hydroxyproline methyl amide. All three derivatives exhibited multiple close intermolecular C-H/O interactions in the crystallographic state, with H⋅⋅⋅O distances as close as 2.3 Å. These observed distances are well below the 2.72 Å sum of the van der Waals radii of H and O, and suggest that these interactions are particularly favorable. In order to generalize these results, we further analyzed the role of C-H/O interactions in all previously crystallized derivatives of these amino acids, and found that all 26 structures exhibited close intermolecular C-H/O interactions. Finally, we analyzed all proline residues in the Cambridge Structural Database of small-molecule crystal structures. We found that the majority of these structures exhibited intermolecular C-H/O interactions at proline C-H bonds, suggesting that C-H/O interactions are an inherent and important mode for recognition of and higher-order assembly at proline residues. Due to steric accessibility and multiple polarized C-H bonds, proline residues are uniquely positioned as sites for binding and recognition via C-H/O interactions., (© 2022 Wiley-VCH GmbH.)

The inhibitor of virus replication (IVR) is an inducible protein that is not virus-target-specific and can be induced by several viruses. The GenBank was interrogated for sequences closely related to the tobacco IVR. Various RNA fragments from tobacco, tomato, and potato and their genomic DNA contained IVR-like sequences. However, IVRs were part of larger proteins encoded by these genomic DNA sequences, which were identified in Arabidopsis as being related to the cyclosome protein designated anaphase-promoting complex 7 (APC7). Sequence analysis of the putative APC7s of nine plant species showed proteins of 558-561 amino acids highly conserved in sequence containing at least six protein-binding elements of 34 amino acids called tetratricopeptide repeats (TPRs), which form helix-turn-helix structures. The structures of Arabidopsis APC7 and the tobacco IVR proteins were modeled using the AlphaFold program and superimposed, showing that IVR had the same structure as the C-terminal 34% of APC7, indicating that IVR was a product of the APC7 gene. Based on the presence of various transcription factor binding sites in the APC7 sequences upstream of the IVR coding sequences, we propose that IVR could be expressed by these APC7 gene sequences involving the transcription factor SHE1.