Toby James Gibson

Pathogens infect hosts by interacting with host proteins and exploiting their functions to their advantage. Short linear motifs, small functional regions within intrinsically disordered protein regions, are common mediators of host-pathogen protein interactions. While motifs have been more extensively studied in viruses and bacteria, the extent to which eukaryotic unicellular parasites use motifs during infection remains poorly explored. Toxoplasma gondii is a widespread intracellular apicomplexan parasite capable of infecting all warm-blooded animals and invading any of their nucleated cells. Toxoplasma ’s secreted proteins are key in interacting with host proteins during infection, making them potential sources for motifs. To study the role of motifs in Toxoplasma gondii infection, we curated 19 known motif instances in Toxoplasma proteins from the scientific literature. To identify more motifs in Toxoplasma- secreted proteins, we developed a computational pipeline that predicts and annotates putative motif matches with structural and functional features. Using this approach, we identified 24,097 motif matches within 295 proteins from secretory organelles. We highlight strategies to further prioritize likely functional motif matches by focusing on integrin motifs, degrons, TRAF6-binding motifs, and 42 confirmed secreted proteins. We subjected peptides containing four predicted TRAF6-binding motifs to experimental validation, supporting the predicted motifs in the Toxoplasma proteins RON10 and GRA15. Our motif predictions provide a valuable resource for generating hypotheses and designing experiments to study infection mechanisms. The characterization of motifs in Toxoplasma will be key to understanding the molecular principles underlying its broad host range and more comprehensive apicomplexan infection strategies. IMPORTANCE: Toxoplasma gondii is a widely distributed intracellular parasite that achieves successful infection by interacting with different host cell proteins. Short linear motifs are small functional modules found in unstructured protein regions and recognized by folded protein domains. Given that unstructured protein regions are a common feature of Toxoplasma ’s proteins, we hypothesize that motifs play important roles during its infection cycle. Here, we highlight the role of motifs during the Toxoplasma host cell invasion cycle through a curated set of motif examples. Through a computational pipeline, we predict thousands of motifs in secreted proteins, outline strategies for working with these predictions, and finally experimentally test peptides containing a motif involved in the innate immune response, successfully supporting the potential binding of two motifs. Our work provides a resource for further motif testing in Toxoplasma proteins, aiming at understanding the molecular mechanisms of the organism’s infection strategies and its broad host range.

Pathogens infect hosts by interacting with host proteins and exploiting their functions to their advantage. Short linear motifs, small functional regions within intrinsically disordered protein regions, are common mediators of host-pathogen protein interactions. While motifs have been more extensively studied in viruses and bacteria, the extent to which eukaryotic unicellular parasites use motifs during infection remains unexplored. Toxoplasma gondii is a widespread intracellular Apicomplexan parasite capable of infecting all warm-blooded animals and invading any of their nucleated cells. Toxoplasma’s secreted proteins are key in interacting with host proteins during infection, making them potential sources for motifs. To highlight the role of motifs in Toxoplasma gondii infection, we curated 21 known motif instances in Toxoplasma proteins from the scientific literature. To identify more motifs in Toxoplasma secreted proteins, we developed a computational pipeline that annotates putative motif matches with structural and functional features. Through this approach, we identified a set of 24,291 motif matches in 295 secreted proteins. We highlight strategies for further prioritisation of likely functional motif matches by focusing on integrin motifs, degrons and TRAF6-binding motifs. We subjected four predicted TRAF6-binding motifs to experimental validation, supporting the predicted motifs in the Toxoplasma proteins RON10 and GRA15. Our motif predictions provide a valuable resource for generating hypotheses and designing experiments to study infection mechanisms. The characterisation of motifs in Toxoplasma will be key to understanding the molecular principles underlying its broad host range and more comprehensive Apicomplexan infection strategies. Importance: Toxoplasma gondii is a widely distributed intracellular parasite that achieves a successful infection by interacting with different host cell proteins. Short linear motifs are small functional modules found in unstructured protein regions and recognised by folded protein domains. Given that unstructured protein regions are a common feature of Toxoplasma’s proteins, we hypothesise that motifs play important roles during its infection cycle. Here, we highlight the role of motifs during the Toxoplasma host cell invasion cycle through a curated set of motif examples. Through a computational pipeline, we predict thousands of motifs in secreted proteins, outline strategies for working with these predictions and finally experimentally test proteins containing a motif involved in the innate immune response, successfully showing the binding of two motifs. Our work provides a resource for further motif testing in Toxoplasma proteins, aiming at understanding the molecular mechanisms of its infection strategies and its broad host range.

Intrinsically disordered protein regions (IDRs) mediate key steps in cellular signaling but the nature of the energy barriers crossed by IDRs upon association and the energetic features of the transition state ensemble (TSE) for binding remain elusive. Short linear motifs (SLiMs) are small functional units found within IDRs that fold upon binding to globular domains. Here, we use the LxCxE SLiM from the human papillomavirus E7 protein (LxCxE_WT) binding to the retinoblastoma (Rb) protein as a minimal model system for an IDR-domain interaction. We combine extensive mutagenesis with electrostatic dissection to gain information on the binding TSE energetics and rule out ground state effects using Far-UV CD and NMR. This approach uncovers strong compensatory energetics whereby stabilizing electrostatic interactions play a key role buffering multiple weakly destabilizing non-native interactions in the binding TSE for LxCxE_WT. Electrostatic buffering may enable a dynamic search for native contacts and fine tuning of the TSE energetics. A global analysis of 177 mutations reveals non-native energetics in the binding TSE of many IDRs, suggesting that electrostatic buffering may be a widespread phenomenon that dictates functional selection for charge content within IDRs.

Structural resolution of protein interactions enables mechanistic and functional studies as well as interpretation of disease variants. However, structural data is still missing for most protein interactions because we lack computational and experimental tools at scale. This is particularly true for interactions mediated by short linear motifs occurring in disordered regions of proteins. We find that AlphaFold-Multimer predicts with high sensitivity but limited specificity structures of domain-motif interactions when using small protein fragments as input. Sensitivity decreased substantially when using long protein fragments or full length proteins. We delineated a protein fragmentation strategy particularly suited for the prediction of domain-motif interfaces and applied it to interactions between human proteins associated with neurodevelopmental disorders. This enabled the prediction of highly confident and likely disease-related novel interfaces, which we further experimentally corroborated for FBXO23-STX1B, STX1B-VAMP2, ESRRG-PSMC5, PEX3-PEX19, PEX3-PEX16, and SNRPB-GIGYF1 providing novel molecular insights for diverse biological processes. Our work highlights exciting perspectives, but also reveals clear limitations and the need for future developments to maximize the power of Alphafold-Multimer for interface predictions.

Short Linear Motifs (SLiMs) are the smallest structural and functional components of modular eukaryotic proteins. They are also the most abundant, especially when considering post-translational modifications. As well as being found throughout the cell as part of regulatory processes, SLiMs are extensively mimicked by intracellular pathogens. At the heart of the Eukaryotic Linear Motif (ELM) Resource is a representative (not comprehensive) database. The ELM entries are created by a growing community of skilled annotators and provide an introduction to linear motif functionality for biomedical researchers. The 2024 ELM update includes 346 novel motif instances in areas ranging from innate immunity to both protein and RNA degradation systems. In total, 39 classes of newly annotated motifs have been added, and another 17 existing entries have been updated in the database. The 2024 ELM release now includes 356 motif classes incorporating 4283 individual motif instances manually curated from 4274 scientific publications and including >700 links to experimentally determined 3D structures. In a recent development, the InterPro protein module resource now also includes ELM data. ELM is available at: http://elm.eu.org.

The SH2-binding phosphotyrosine class of short linear motifs (SLiMs) are key conditional regulatory elements, particularly in signaling protein complexes beneath the cell’s plasma membrane. In addition to transmitting cellular signaling information, they can also play roles in cellular hijack by invasive pathogens. Researchers can take advantage of bioinformatics tools and resources to predict the motifs at conserved phosphotyrosine residues in regions of intrinsically disordered protein. A candidate SH2-binding motif can be established and assigned to one or more of the SH2 domain subgroups. It is, however, not so straightforward to predict which SH2 domains are capable of binding the given candidate. This is largely due to the cooperative nature of the binding amino acids which enables poorer binding residues to be tolerated when the other residues are optimal. High-throughput peptide arrays are powerful tools used to derive SH2 domain-binding specificity, but they are unable to capture these cooperative effects and also suffer from other shortcomings. Tissue and cell type expression can help to restrict the list of available interactors: for example, some well-studied SH2 domain proteins are only present in the immune cell lineages. In this article, we provide a table of motif patterns and four bioinformatics strategies that introduce a range of tools that can be used in motif hunting in cellular and pathogen proteins. Experimental followup is essential to determine which SH2 domain/motif-containing proteins are the actual functional partners. Key words SH2 domain Short linear motif (SLiM) Regular expression Binding specificity Phosphotyrosine Cell signaling Pathogen hijack

The pathogenic tropical flagellates Leishmania belong to an early-branching eukaryotic lineage (Kinetoplastida) with several unique features. Here, we explore three ancient protein targeting linear motif systems and their receptors and demonstrate how they resemble or differ from other eukaryotic organisms, including their hosts. Secretory signal peptides, endoplasmic reticulum (ER) retention motifs (KDEL motifs), and autophagy signals (motifs interacting with ATG8 family members) are essential components of cellular life. Although expected to be conserved, we observe that all three systems show a varying degree of divergence from the eukaryotic version observed in animals, plants, or fungi. We not only describe their behavior but also build predictive models that allow the prediction of localization or function for several proteins in Leishmania species for the first time. Several of these critical protein-protein interactions could serve as targets of selective antimicrobial agents against Leishmaniasis due to their divergence from the host.

DisProt is the primary repository of Intrinsically Disordered Proteins (IDPs). This database is manually curated and the annotations there have strong experimental support. Currently, DisProt contains a relatively small number of proteins highlighting the importance of transferring annotations regarding verified disorder state and corresponding functions to homologous proteins in other species. In such a way, providing them with highly valuable information to better understand their biological roles. While the principles and practicalities of homology transfer are well‐established for globular proteins, these are largely lacking for disordered proteins. We used DisProt to evaluate the transferability of the annotation terms to orthologous proteins. For each protein, we looked for their orthologs, with the assumption that they will have a similar function. Then, for each protein and their orthologs, we made multiple sequence alignments (MSAs). Disordered sequences are fast evolving and can be hard to align, therefore, we implemented alignment quality control steps ensuring robust alignments before mapping the annotations. We have designed a pipeline to obtain good‐quality MSAs and to transfer annotations from any protein to their orthologs. Applying the pipeline to DisProt proteins, from the 1731 entries with 5623 annotations, we can reach 97,555 orthologs and transfer a total of 301,190 terms by homology. We also provide a web server for consulting the results of DisProt proteins and execute the pipeline for any other protein. The server Homology Transfer IDP (HoTIDP) is accessible at http://hotidp.leloir.org.ar.

Leishmaniasis is a detrimental disease causing serious changes in quality of life and some forms lead to death. The disease is spread by the parasite Leishmania transmitted by sandfly vectors and their primary hosts are vertebrates including humans. The pathogen penetrates host cells and secretes proteins (the secretome) to repurpose cells for pathogen growth and to alter cell signaling via host-pathogen Protein-Protein Interactions (PPIs). Here we present LeishMANIAdb, a database specifically designed to investigate how Leishmania virulence factors may interfere with host proteins. Since the secretomes of different Leishmania species are only partially characterized, we collected various experimental evidence and used computational predictions to identify Leishmania secreted proteins to generate a user-friendly unified web resource allowing users to access all information available on experimental and predicted secretomes. In addition, we manually annotated host-pathogen interactions of 211 proteins, and the localization/function of 3764 transmembrane (TM) proteins of different Leishmania species. We also enriched all proteins with automatic structural and functional predictions that can provide new insights in the molecular mechanisms of infection. Our database, available at https://leishmaniadb.ttk.hu may provide novel insights into Leishmania host-pathogen interactions and help to identify new therapeutic targets for this neglected disease.

Background DisProt is the primary repository of Intrinsically Disordered Proteins. This database is manually curated and the annotations there have strong experimental support. Currently DisProt contains a relatively small number of proteins highlighting the importance of transferring verified disorder and other annotations, in such a way as to increase the number of proteins that could benefit from this valuable information. While the principles and practicalities of homology transfer are well-established for globular proteins, these are largely lacking for disordered proteins. Methods We used DisProt to evaluate the transferability of the annotation terms to orthologous proteins. For each protein, we looked for their orthologs, with the assumption that they will have a similar function. Then, for each protein and their orthologs we made multiple sequence alignments (MSAs). Global and regional quality of the MSAs was evaluated with the NorMD score. Results We have designed a pipeline to obtain good quality MSAs and to transfer annotations from any protein to their orthologs. Applying the pipeline to DisProt proteins, from the 1931 entries with 5,623 annotations we can reach 97,555 orthologs and transfer a total of 301,190 terms by homology. We also provide a web server for consulting the results of DisProt proteins and execute the pipeline for any other protein. The server Homology Transfer IDP (HoTIDP) is accessible at http://hotidp.leloir.org.ar.