ELM-the Eukaryotic Linear Motif resource-2024 update.

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.

Evolution of SLiM-mediated hijack functions in intrinsically disordered viral proteins.

Viruses and their hosts are involved in an 'arms race' where they continually evolve mechanisms to overcome each other. It has long been proposed that intrinsic disorder provides a substrate for the evolution of viral hijack functions and that short linear motifs (SLiMs) are important players in this process. Here, we review evidence in support of this tenet from two model systems: the papillomavirus E7 protein and the adenovirus E1A protein. Phylogenetic reconstructions reveal that SLiMs appear and disappear multiple times across evolution, providing evidence of convergent evolution within individual viral phylogenies. Multiple functionally related SLiMs show strong coevolution signals that persist across long distances in the primary sequence and occur in unrelated viral proteins. Moreover, changes in SLiMs are associated with changes in phenotypic traits such as host range and tropism. Tracking viral evolutionary events reveals that host switch events are associated with the loss of several SLiMs, suggesting that SLiMs are under functional selection and that changes in SLiMs support viral adaptation. Fine-tuning of viral SLiM sequences can improve affinity, allowing them to outcompete host counterparts. However, viral SLiMs are not always competitive by themselves, and tethering of two suboptimal SLiMs by a disordered linker may instead enable viral hijack. Coevolution between the SLiMs and the linker indicates that the evolution of disordered regions may be more constrained than previously thought. In summary, experimental and computational studies support a role for SLiMs and intrinsic disorder in viral hijack functions and in viral adaptive evolution.

Conformational buffering underlies functional selection in intrinsically disordered protein regions.

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

Host diversification is concurrent with linear motif evolution in a Mastadenovirus hub protein.

Over one hundred Mastadenovirus types infect seven orders of mammals. Virus-host coevolution may involve cospeciation, duplication, host switch and partial extinction events. We reconstruct Mastadenovirus diversification, finding that while cospeciation is dominant, the other three events are also common in Mastadenovirus evolution. Linear motifs are fast-evolving protein functional elements and key mediators of virus-host interactions, thus likely to partake in adaptive viral evolution. We study the evolution of eleven linear motifs in the Mastadenovirus E1A protein, a hub of virus-host protein-protein interactions, in the context of host diversification. The reconstruction of linear motif gain and loss events shows fast linear motif turnover, corresponding a virus-host protein-protein interaction turnover orders of magnitude faster than in model host proteomes. Evolution of E1A linear motifs is coupled, indicating functional coordination at the protein scale, yet presents motif-specific patterns suggestive of convergent evolution. We report a pervasive association between Mastadenovirus host diversification events and the evolution of E1A linear motifs. Eight of 17 host switches associate with the gain of one linear motif and the loss of four different linear motifs, while five of nine partial extinctions associate with the loss of one linear motif. The specific changes in E1A linear motifs during a host switch or a partial extinction suggest that changes in the host molecular environment lead to modulation of the interactions with the retinoblastoma protein and host transcriptional regulators. Altogether, changes in the linear motif repertoire of a viral hub protein are associated with adaptive evolution events during Mastadenovirus evolution.

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.

Predicting the effect of disordered linkers on effective concentrations and avidity with the "Ceff calculator" app.

Linkers are crucial to the functions of multidomain proteins as they couple functional units to encode regulation such as auto-inhibition, enzyme targeting or tuning of interaction strength. A linker changes reactions from bimolecular to unimolecular, and the equilibrium and kinetics is thus determined by the properties of the linker rather than concentrations. We present a theoretical workflow for estimating the functional consequences of tethering by a linker. We discuss how to: (1) Identify flexible linkers from sequence. (2) Model the end-to-end distance distribution for a flexible linker using a worm-like chain. (3) Estimate the effective concentration of a ligand tethered by a flexible linker. (4) Calculate the decrease in binding affinity caused by auto-inhibition. (5) Calculate the expected avidity enhancement of a bivalent interaction from effective concentration. The worm-like chain modeling is available through a web application called the "Ceff calculator" (http://ceffapp.chemeslab.org), which will allow user-friendly prediction of experimentally inaccessible parameters.

ELM-the eukaryotic linear motif resource in 2020.

The eukaryotic linear motif (ELM) resource is a repository of manually curated experimentally validated short linear motifs (SLiMs). Since the initial release almost 20 years ago, ELM has become an indispensable resource for the molecular biology community for investigating functional regions in many proteins. In this update, we have added 21 novel motif classes, made major revisions to 12 motif classes and added >400 new instances mostly focused on DNA damage, the cytoskeleton, SH2-binding phosphotyrosine motifs and motif mimicry by pathogenic bacterial effector proteins. The current release of the ELM database contains 289 motif classes and 3523 individual protein motif instances manually curated from 3467 scientific publications. ELM is available at: http://elm.eu.org.

Interplay between sequence, structure and linear motifs in the adenovirus E1A hub protein.

E1A is the main transforming protein in mastadenoviruses. This work uses bioinformatics to extrapolate experimental knowledge from Human adenovirus serotype 5 and 12 E1A proteins to all known serotypes. A conserved domain architecture with a high degree of intrinsic disorder acts as a scaffold for multiple linear motifs with variable occurrence mediating the interaction with over fifty host proteins. While linear motifs contribute strongly to sequence conservation within intrinsically disordered E1A regions, motif repertoires can deviate significantly from those found in prototypical serotypes. Close to one hundred predicted residue-residue contacts suggest the presence of stable structure in the CR3 domain and of specific conformational ensembles involving both short- and long-range intramolecular interactions. Our computational results suggest that E1A sequence conservation and co-evolution reflect the evolutionary pressure to maintain a mainly disordered, yet non-random conformation harboring a high number of binding motifs that mediate viral hijacking of the cell machinery.

The eukaryotic linear motif resource ELM: 10 years and counting.

The eukaryotic linear motif (ELM http://elm.eu.org) resource is a hub for collecting, classifying and curating information about short linear motifs (SLiMs). For >10 years, this resource has provided the scientific community with a freely accessible guide to the biology and function of linear motifs. The current version of ELM contains ∼200 different motif classes with over 2400 experimentally validated instances manually curated from >2000 scientific publications. Furthermore, detailed information about motif-mediated interactions has been annotated and made available in standard exchange formats. Where appropriate, links are provided to resources such as switches.elm.eu.org and KEGG pathways.

Sequence evolution of the intrinsically disordered and globular domains of a model viral oncoprotein.

In the present work, we have used the papillomavirus E7 oncoprotein to pursue structure-function and evolutionary studies that take into account intrinsic disorder and the conformational diversity of globular domains. The intrinsically disordered (E7N) and globular (E7C) domains of E7 show similar degrees of conservation and co-evolution. We found that E7N can be described in terms of conserved and coevolving linear motifs separated by variable linkers, while sequence evolution of E7C is compatible with the known homodimeric structure yet suggests other activities for the domain. Within E7N, inter-residue relationships such as residue co-evolution and restricted intermotif distances map functional coupling and co-occurrence of linear motifs that evolve in a coordinate manner. Within E7C, additional cysteine residues proximal to the zinc-binding site may allow redox regulation of E7 function. Moreover, we describe a conserved binding site for disordered domains on the surface of E7C and suggest a putative target linear motif. Both homodimerization and peptide binding activities of E7C are also present in the distantly related host PHD domains, showing that these two proteins share not only structural homology but also functional similarities, and strengthening the view that they evolved from a common ancestor. Finally, we integrate the multiple activities and conformations of E7 into a hierarchy of structure-function relationships.

Evolution of linear motifs within the papillomavirus E7 oncoprotein.

Many protein functions can be traced to linear sequence motifs of less than five residues, which are often found within intrinsically disordered domains. In spite of their prevalence, their role in protein evolution is only beginning to be understood. The study of papillomaviruses has provided many insights on the evolution of protein structure and function. We have chosen the papillomavirus E7 oncoprotein as a model system for the evolution of functional linear motifs. The multiple functions of E7 proteins from paradigmatic papillomavirus types can be explained to a large extent in terms of five linear motifs within the intrinsically disordered N-terminal domain and two linear motifs within the globular homodimeric C-terminal domain. We examined the motif inventory of E7 proteins from over 200 known papillomavirus types and found that the motifs reported for paradigmatic papillomavirus types are absent from many uncharacterized E7 proteins. Several motif pairs occur more often than expected, suggesting that linear motifs may evolve and function in a cooperative manner. The E7 linear motifs have appeared or disappeared multiple times during papillomavirus evolution, confirming the evolutionary plasticity of short functional sequences. Four of the motifs appeared several times during papillomavirus evolution, providing direct evidence for convergent evolution. Interestingly, the evolution pattern of a motif is independent of its location in a globular or disordered domain. The correlation between the presence of some motifs and virus host specificity and tissue tropism suggests that linear motifs play a role in the adaptive evolution of papillomaviruses.