Biology:Short linear motif

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The human papilloma virus E7 oncoprotein mimic of the LxCxE motif (red) bound to the host retinoblastoma protein (dark grey) (PDB: 1gux​)

In molecular biology short linear motifs (SLiMs), linear motifs or minimotifs are short stretches of protein sequence that mediate protein–protein interaction.[1][2]

The first definition was given by Tim Hunt:[3]

"The sequences of many proteins contain short, conserved motifs that are involved in recognition and targeting activities, often separate from other functional properties of the molecule in which they occur. These motifs are linear, in the sense that three-dimensional organization is not required to bring distant segments of the molecule together to make the recognizable unit. The conservation of these motifs varies: some are highly conserved while others, for example, allow substitutions that retain only a certain pattern of charge across the motif."

Attributes

SLiMs are generally situated in intrinsically disordered regions [4] (over 80% of known SLiMs), however, upon interaction with a structured partner secondary structure is often induced. The majority of annotated SLiMs consist of 3 to 11 contiguous amino acids, with an average of just over 6 residues. However, only few hotspot residues (on average 1 hotspot for each 3 residues in the motif) contribute the majority of the free energy of binding and determine most of the affinity and specificity of the interaction. Although most motifs have no positional preference, several of them are required to be localized at the protein termini in order to be functional.[5][6] The key defining attribute of SLiMs, having a limited number of residues that directly contact the binding partner, has two major consequences. First, only few or even a single mutation can result in the generation of a functional motif, with further mutations of flanking residues allowing tuning affinity and specificity.[7] This results in SLiMs having an increased propensity to evolve convergently, which facilitates their proliferation, as is evidenced by their conservation and increased incidence in higher Eukaryotes.[8] It has been hypothesized that this might increase and restructure the connectivity of the interactome. Second, SLiMs have relatively low affinity for their interaction partners (generally between 1 and 150 μM), which makes these interactions transient and reversible, and thus ideal to mediate dynamic processes such as cell signaling. In addition, this means that these interactions can be easily modulated by post-translational modifications that change the structural and physicochemical properties of the motif. Also, regions of high functional density can mediate molecular switching by means of overlapping motifs (e.g. the C-terminal tails of integrin beta subunits), or they can allow high avidity interactions by multiple low affinity motifs (e.g. multiple AP2-binding motifs in Eps15).[6][9][10]

Function

SLiM functions in almost every pathway due to their critical role in regulatory function, protein-protein interaction and signal transduction. SLiM act as interaction modules that are recognised by additional biomolecules. The majority of known interaction partners of SLiMs are globular protein domains, though, SLiMs that recognise other intrinsically disordered regions, RNA and lipids have also been characterised. SLiMs can be broadly split into two high level classes, modification sites and ligand binding sites.

Modification sites
Modification sites SLiMs encompass sites with intrinsic specificity determinant that are recognised and modified by the active site of a catalytic domain of an enzyme. These SLiMs include many classical post translational modification sites (PTMs), proteolytic cleavage sites recognised by proteases and bonds recognised by isomerases.

  • Moiety addition – SLiMs are often targeted for the addition of a small chemical groups (e.g. Phosphorylation), proteins (e.g. SUMOylation) or other moieties (e.g. post translational moiety addition).
  • Proteolytic cleavage -SLiMs can act as recognition sites of endo-peptidases resulting in the irreversible cleavage of the peptide at the SLiM.
  • Structural modifications – SLiMs can be recognised by isomerases resulting in the cis-trans isomerisation of the peptide backbone.

Ligand binding sites
Ligand binding site SLiMs recruit binding partners to the SLiM containing proteins, often mediating transient interactions, or acting co-operatively to produce more stable complexes. Ligand SLiMs are often central to the formation of dynamic multi-protein complexes, however, they more commonly mediate regulatory interactions that control the stability, localisation or modification state of a protein.[11]

  • Complex formation – Ligand SLiMs often function as simple interfaces that recruit proteins to multi-protein complexes (e.g. the Retinoblastoma-binding LxCxE motif) or act as aggregators in scaffold proteins (e.g. SH3 domain-binding proline-rich sequences).
  • Localisation – A large number of SLiMs act as zipcodes that are recognized by the cellular transport machinery mediating the relocalisation of the containing protein to the correct sub-cellular compartment (e.g. Nuclear localisation signals (NLSs) and Nuclear export signals (NESs))
  • Modification state – Many classes of ligand SLiMs recruit enzymes to their substrate by binding to sites that are distinct from the enzyme's active site. These site, known as docking motifs, act as additional specificity determinants for these enzymes and decrease the likelihood of off-target modification events.
  • Stability – A subset of docking motifs recruit E3 ubiquitin ligase to their substrates. The resulting polyubiquitination targets the substrate for proteosomal destruction.

Role in disease

Disordered protein elements like SLiMs are frequently found in factors that regulate gene expression.[11] As a result, several diseases have been linked to mutations that alter key SLiM-mediated functions. For instance, one cause of Noonan Syndrome is a mutation in the protein Raf-1 which abrogates the interaction with 14-3-3 proteins mediated by corresponding short linear motifs and thereby deregulate the Raf-1 kinase activity.[12] Usher's Syndrome is the most frequent cause of hereditary deaf-blindness in humans[13] and can be caused by mutations in either PDZ domains in Harmonin or the corresponding PDZ interaction motifs in the SANS protein.[14] Finally, Liddle's Syndrome has been implicated with autosomal dominant activating mutations in the WW interaction motif in the β-(SCNNB_HUMA) and γ-(SCNNG_HUMA) subunits of the Epithelial sodium channel ENaC.[15] These mutations abrogate the binding to the ubiquitin ligase NEDD4, thereby inhibiting channel degradation and prolonging the half-life of ENaC, ultimately resulting in increased Na+ reabsorption, plasma volume extension and hypertension.[16]

Viruses often mimic human SLiMs to hijack and disrupt a host's cellular machinery,[17][18][11] thereby adding functionality to their compact genomes without necessitating new virally encoded proteins. In fact, many motifs were originally discovered in viruses, such as the Retinoblastoma binding LxCxE motif and the UEV domain binding PTAP late domain. The short generation times and high mutation rates of viruses, in association with natural selection, has led to multiple examples of mimicry of host SLiMs in every step of the viral life cycle (Src binding motif PxxP in Nef modulates replication, WW domain binding PPxY mediates budding in Ebola virus, A Dynein Light Chain binding motif in Rabies virus is vital for host infection). The extent of human SLiM mimicry is surprising with many viral proteins containing several functional SLiMs, for example, the Adenovirus protein E1A.

Pathogenic bacteria also mimic host motifs (as well as having their own motifs), however, not to the same extent as the obligate parasite viruses. E. Coli injects a protein, EspF(U), that mimics an autoinhibitory element of N-WASP into the host cell to activate actin-nucleating factors WASP.[19] The KDEL motif of the bacteria encoded cholera toxin mediates cell entry of the cholera toxin.[20]

MDM2 SWIB domain-binding motif mimic drug Nutlin bound to MDM2(PDB: 3lbk​)

Potential as leads for drug design

Linear motif mediated protein-protein interactions have shown promise in recent years as novel drug targets.[21] Success stories include the MDM2 motif analog Nutlin-3 and integrin targeting RGD-mimetic Cilengitide: Nutlin-3 antagonises the interaction of MDM2's SWIB domain with p53 thus stabilising p53 and inducing senescence in cancer cells.[22] Cilengitide inhibits integrin-dependent signaling, causing the disassembly of cytoskeleton, cellular detachment and the induction of apoptosis in endothelial and glioma cells.[23][24] In addition, peptides targeting the Grb2 and Crk SH2/ SH3 adaptor domains are also under investigation.[25][26]

There are at present no drugs on the market specially targeting phosphorylation sites, however, a number of drugs target the kinase domain. This tactic has shown promise in the treatments of various forms of cancer.[18] For example, Stutnet® is a receptor tyrosine kinase (RTK) inhibitor for treating gastrointestinal cancer, Gleevec® specially targets bcr-abl and Sprycel® is a broad-based tyrosine kinase inhibitor whose targets include Bcr-Abl and Src. Cleavage is another process directed by motif recognition with the proteases responsible for cleavage a good drug target. For example, Tritace®, Vasotec®, Accupril®, and Lotensin® are substrate mimetic Angiotensin converting enzymes inhibitors. Other drugs that target post-translational modifications include Zovirax®, an antiviral myristoylation inhibitor and Farnysyl Transferase inhibitors that block the lipidation modification to a CAAX-box motif.

Recommended further reading:[18][27]

Computational motif resources

Databases

SLiMs are usually described by regular expressions in the motif literature with the important residues defined based on a combination of experimental, structural and evolutionary evidence. However, high throughput screening such as phage display has seen a large increase in the available information for many motifs classes allowing them to be described with sequence logos.[28] Several diverse repositories currently curate the available motif data. In terms of scope, the Eukaryotic Linear Motif resource (ELM)[29] and MiniMotif Miner (MnM)[30] represent the two largest motif databases as they attempt to capture all motifs from the available literature. Several more specific and specialised databases also exist, PepCyber[31] and ScanSite[32] focus on smaller subsets of motifs, phosphopeptide binding and important signaling domains respectively. PDZBase[33] focuses solely on PDZ domain ligands. MEROPS[34] and CutDB[35] curate available proteolytic event data including protease specificity and cleavage sites. There has been a large increase in the number of publications describing motif mediated interactions over past decade and as a result a large amount of the available literature remains to be curated. Recent work has created the tool MiMosa[36] to expedite the annotation process and encourage semantically robust motif descriptions.[37]

Discovery tools

SLiMs are short and degenerate and as a result the proteome is littered with stochastically occurring peptides that resemble functional motifs. The biologically relevant cellular partners can easily distinguish functional motifs, however computational tools have yet to reach a level of sophistication where motif discovery can be accomplished with high success rates.

Motif discovery tools can be split into two major categories, discovery of novel instance of known functional motifs class and discovery of functional motifs class, however, they all use a limited and overlapping set of attributes to discriminate true and false positives. The main discrimatory attributes used in motif discovery are:

  • Accessibility – the motif must be accessible for the binding partner. Intrinsic disorder prediction tools (such as IUPred or GlobPlot), domain databases (such as Pfam and SMART) and experimentally derived structural data (from sources such as PDB) can be used to check the accessibility of predicted motif instances.
  • Conservation – the conservation of a motif correlates strongly with functionality and many experimental motifs are seen as islands of strong constraint in regions of weak conservation. Alignment of homologous proteins can be used to calculate conservation metric for a motif.
  • Physicochemical properties – Certain intrinsic properties of residues or stretches of amino acids are strong discriminators of functionality, for example, the propensity of a region of disorder to undergo a disorder to order transition.
  • Enrichment in groupings of similar proteins – Motif often evolve convergently to carry out similar tasks in different proteins such as mediating binding to a specific partner or targeting proteins to a particular subcellular localisation. Often in such cases these grouping the motif occurs more often than is expected by chance and can be detected by searching for enriched motifs.

Novel functional motifs instances

The Eukaryotic Linear Motif resource (ELM)[29] and MiniMotif Miner (MnM)[30] both provide servers to search for novel instance of known functional motifs in protein sequences. SLiMSearch allows similar searches on a proteome-wide scale.[38]

Novel functional motifs class

More recently computational methods have been developed that can identify new Short Linear Motifs de novo.[39] Interactome-based tools rely on identifying a set of proteins that are likely to share a common function, such as binding the same protein or being cleaved by the same peptidase. Two examples of such software are DILIMOT and SLiMFinder.[40][41] Anchor and α-MoRF-Pred use physicochemical properties to search for motif-like peptides in disordered regions (termed MoRFs, among others). ANCHOR[42] identifies stretches of intrinsically disordered regions that cannot form favorable intrachain interactions to fold without additional stabilising energy contributed by a globular interaction partner. α-MoRF-Pred[43] uses the inherent propensity of many SLiM to undergo a disorder to order transition upon binding to discover α-helical forming stretches within disordered regions. MoRFPred[44] and MoRFchibi SYSTEM[45][46][47] are SVM based predictors which utilize multiple features including local sequence physicochemical properties, long stretches of disordered regions and conservation in their predictions. SLiMPred[48] is neural network–based method for the de novo discovery of SLiMs from the protein sequence. Information about the structural context of the motif (predicted secondary structure, structural motifs, solvent accessibility, and disorder) are used during the predictive process. Importantly, no previous knowledge about the protein (i.e., no evolutionary or experimental information) is required.

References

  1. ↑ "Understanding eukaryotic linear motifs and their role in cell signaling and regulation". Frontiers in Bioscience 13 (13): 6580–603. May 2008. doi:10.2741/3175. PMID 18508681. 
  2. ↑ "Peptides mediating interaction networks: new leads at last". Current Opinion in Biotechnology 17 (5): 465–71. October 2006. doi:10.1016/j.copbio.2006.08.002. PMID 16962311. 
  3. ↑ "Peptide sequences that target cytosolic proteins for lysosomal proteolysis". Trends in Biochemical Sciences 15 (8): 305–9. August 1990. doi:10.1016/0968-0004(90)90019-8. PMID 2204156. 
  4. ↑ "Short Linear Motifs recognized by SH2, SH3 and Ser/Thr Kinase domains are conserved in disordered protein regions". BMC Genomics 9 (Suppl 2): S26. September 2008. doi:10.1186/1471-2164-9-S2-S26. PMID 18831792. 
  5. ↑ "The structural basis of peptide-protein binding strategies". Structure 18 (2): 188–99. February 2010. doi:10.1016/j.str.2009.11.012. PMID 20159464. 
  6. ↑ 6.0 6.1 "Attributes of short linear motifs". Molecular BioSystems 8 (1): 268–81. January 2012. doi:10.1039/c1mb05231d. PMID 21909575. 
  7. ↑ "Short linear motifs - ex nihilo evolution of protein regulation". Cell Communication and Signaling 13 (1): 43. November 2015. doi:10.1186/s12964-015-0120-z. PMID 26589632. 
  8. ↑ "The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains". BMC Genomics 9: 452. October 2008. doi:10.1186/1471-2164-9-452. PMID 18828911. 
  9. ↑ "Linear motifs: evolutionary interaction switches". FEBS Letters 579 (15): 3342–5. June 2005. doi:10.1016/j.febslet.2005.04.005. PMID 15943979. 
  10. ↑ "Cell regulation: determined to signal discrete cooperation". Trends in Biochemical Sciences 34 (10): 471–82. October 2009. doi:10.1016/j.tibs.2009.06.007. PMID 19744855. 
  11. ↑ 11.0 11.1 11.2 Cermakova, Katerina; Hodges, H. Courtney (2023-02-06). "Interaction modules that impart specificity to disordered protein". Trends in Biochemical Sciences 48 (5): S0968–0004(23)00008–7. doi:10.1016/j.tibs.2023.01.004. ISSN 0968-0004. PMID 36754681. 
  12. ↑ "Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy". Nature Genetics 39 (8): 1007–12. August 2007. doi:10.1038/ng2073. PMID 17603483. 
  13. ↑ "Molecular genetics of Usher syndrome". Cellular and Molecular Life Sciences 56 (3–4): 258–67. October 1999. doi:10.1007/s000180050427. PMID 11212353. 
  14. ↑ "A novel D458V mutation in the SANS PDZ binding motif causes atypical Usher syndrome". Journal of Molecular Medicine 83 (12): 1025–32. December 2005. doi:10.1007/s00109-005-0719-4. PMID 16283141. 
  15. ↑ "Liddle syndrome: an autosomal dominant form of human hypertension". Kidney International 53 (1): 18–24. January 1998. doi:10.1046/j.1523-1755.1998.00728.x. PMID 9452995. 
  16. ↑ "Liddle's syndrome caused by a novel mutation in the proline-rich PY motif of the epithelial sodium channel beta-subunit". The Journal of Clinical Endocrinology and Metabolism 90 (1): 340–4. January 2005. doi:10.1210/jc.2004-1027. PMID 15483078. 
  17. ↑ "How viruses hijack cell regulation". Trends in Biochemical Sciences 36 (3): 159–69. March 2011. doi:10.1016/j.tibs.2010.10.002. PMID 21146412. 
  18. ↑ 18.0 18.1 18.2 "Viral infection and human disease--insights from minimotifs". Frontiers in Bioscience 13 (13): 6455–71. May 2008. doi:10.2741/3166. PMID 18508672. 
  19. ↑ "The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency". Nature 454 (7207): 1005–8. August 2008. doi:10.1038/nature07170. PMID 18650806. Bibcode: 2008Natur.454.1005S. 
  20. ↑ "Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL". The Journal of Cell Biology 131 (4): 951–62. November 1995. doi:10.1083/jcb.131.4.951. PMID 7490296. 
  21. ↑ "Reaching for high-hanging fruit in drug discovery at protein-protein interfaces". Nature 450 (7172): 1001–9. December 2007. doi:10.1038/nature06526. PMID 18075579. Bibcode: 2007Natur.450.1001W. 
  22. ↑ "In vivo activation of the p53 pathway by small-molecule antagonists of MDM2". Science 303 (5659): 844–8. February 2004. doi:10.1126/science.1092472. PMID 14704432. Bibcode: 2004Sci...303..844V. 
  23. ↑ "Nanomolar small molecule inhibitors for alphav(beta)6, alphav(beta)5, and alphav(beta)3 integrins". Journal of Medicinal Chemistry 45 (5): 1045–51. February 2002. doi:10.1021/jm0102598. PMID 11855984. 
  24. ↑ "Cilengitide induces cellular detachment and apoptosis in endothelial and glioma cells mediated by inhibition of FAK/src/AKT pathway". Journal of Experimental & Clinical Cancer Research 27 (1): 86. December 2008. doi:10.1186/1756-9966-27-86. PMID 19114005. 
  25. ↑ "Grb2-SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel". International Journal of Cancer 121 (2): 407–15. July 2007. doi:10.1002/ijc.22674. PMID 17372910. 
  26. ↑ "Potential disease targets for drugs that disrupt protein-- protein interactions of Grb2 and Crk family adaptors". Current Pharmaceutical Design 12 (5): 529–48. 2006. doi:10.2174/138161206775474369. PMID 16472145. 
  27. ↑ "Intrinsically disordered proteins are potential drug targets". Current Opinion in Chemical Biology 14 (4): 481–8. August 2010. doi:10.1016/j.cbpa.2010.06.169. PMID 20598937. 
  28. ↑ "Profile-based short linear protein motif discovery". BMC Bioinformatics 13: 104. May 2012. doi:10.1186/1471-2105-13-104. PMID 22607209. 
  29. ↑ 29.0 29.1 "ELM: the status of the 2010 eukaryotic linear motif resource". Nucleic Acids Research 38 (Database issue): D167-80. January 2010. doi:10.1093/nar/gkp1016. PMID 19920119. 
  30. ↑ 30.0 30.1 "Minimotif miner 2nd release: a database and web system for motif search". Nucleic Acids Research 37 (Database issue): D185-90. January 2009. doi:10.1093/nar/gkn865. PMID 18978024. 
  31. ↑ "PepCyber:P~PEP: a database of human protein protein interactions mediated by phosphoprotein-binding domains". Nucleic Acids Research 36 (Database issue): D679-83. January 2008. doi:10.1093/nar/gkm854. PMID 18160410. 
  32. ↑ "Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs". Nucleic Acids Research 31 (13): 3635–41. July 2003. doi:10.1093/nar/gkg584. PMID 12824383. 
  33. ↑ "PDZBase: a protein-protein interaction database for PDZ-domains". Bioinformatics 21 (6): 827–8. March 2005. doi:10.1093/bioinformatics/bti098. PMID 15513994. 
  34. ↑ "MEROPS: the peptidase database". Nucleic Acids Research 38 (Database issue): D227-33. January 2010. doi:10.1093/nar/gkp971. PMID 19892822. 
  35. ↑ "CutDB: a proteolytic event database". Nucleic Acids Research 35 (Database issue): D546-9. January 2007. doi:10.1093/nar/gkl813. PMID 17142225. 
  36. ↑ "MimoSA: a system for minimotif annotation". BMC Bioinformatics 11: 328. June 2010. doi:10.1186/1471-2105-11-328. PMID 20565705. 
  37. ↑ "Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis". The EMBO Journal 23 (22): 4371–83. November 2004. doi:10.1038/sj.emboj.7600445. PMID 15496985. 
  38. ↑ "SLiMSearch 2.0: biological context for short linear motifs in proteins". Nucleic Acids Research 39 (Web Server issue): W56-60. July 2011. doi:10.1093/nar/gkr402. PMID 21622654. 
  39. ↑ "SLiM on Diet: finding short linear motifs on domain interaction interfaces in Protein Data Bank". Bioinformatics 26 (8): 1036–42. April 2010. doi:10.1093/bioinformatics/btq065. PMID 20167627. 
  40. ↑ "DILIMOT: discovery of linear motifs in proteins". Nucleic Acids Research 34 (Web Server issue): W350-5. July 2006. doi:10.1093/nar/gkl159. PMID 16845024. 
  41. ↑ "SLiMFinder: a web server to find novel, significantly over-represented, short protein motifs". Nucleic Acids Research 38 (Web Server issue): W534-9. July 2010. doi:10.1093/nar/gkq440. PMID 20497999. 
  42. ↑ Casadio, Rita, ed (May 2009). "Prediction of protein binding regions in disordered proteins". PLOS Computational Biology 5 (5): e1000376. doi:10.1371/journal.pcbi.1000376. PMID 19412530. Bibcode: 2009PLSCB...5E0376M. 
  43. ↑ "Mining alpha-helix-forming molecular recognition features with cross species sequence alignments". Biochemistry 46 (47): 13468–77. November 2007. doi:10.1021/bi7012273. PMID 17973494. 
  44. ↑ "MoRFpred, a computational tool for sequence-based prediction and characterization of short disorder-to-order transitioning binding regions in proteins". Bioinformatics 28 (12): i75-83. June 2012. doi:10.1093/bioinformatics/bts209. PMID 22689782. 
  45. ↑ "Computational identification of MoRFs in protein sequences". Bioinformatics 31 (11): 1738–44. June 2015. doi:10.1093/bioinformatics/btv060. PMID 25637562. 
  46. ↑ "Computational Identification of MoRFs in Protein Sequences Using Hierarchical Application of Bayes Rule". PLOS ONE 10 (10): e0141603. 30 October 2015. doi:10.1371/journal.pone.0141603. PMID 26517836. Bibcode: 2015PLoSO..1041603M. 
  47. ↑ "MoRFchibi SYSTEM: software tools for the identification of MoRFs in protein sequences". Nucleic Acids Research 44 (W1): W488-93. July 2016. doi:10.1093/nar/gkw409. PMID 27174932. 
  48. ↑ "Prediction of short linear protein binding regions". Journal of Molecular Biology 415 (1): 193–204. January 2012. doi:10.1016/j.jmb.2011.10.025. PMID 22079048. 

External links

SLiM databases

SLiM discovery tools



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