SLiMPrints: conservation-based discovery of functional motif fingerprints in intrinsically disordered protein regions - PubMed (original) (raw)
. 2012 Nov;40(21):10628-41.
doi: 10.1093/nar/gks854. Epub 2012 Sep 12.
Affiliations
- PMID: 22977176
- PMCID: PMC3510515
- DOI: 10.1093/nar/gks854
SLiMPrints: conservation-based discovery of functional motif fingerprints in intrinsically disordered protein regions
Norman E Davey et al. Nucleic Acids Res. 2012 Nov.
Abstract
Large portions of higher eukaryotic proteomes are intrinsically disordered, and abundant evidence suggests that these unstructured regions of proteins are rich in regulatory interaction interfaces. A major class of disordered interaction interfaces are the compact and degenerate modules known as short linear motifs (SLiMs). As a result of the difficulties associated with the experimental identification and validation of SLiMs, our understanding of these modules is limited, advocating the use of computational methods to focus experimental discovery. This article evaluates the use of evolutionary conservation as a discriminatory technique for motif discovery. A statistical framework is introduced to assess the significance of relatively conserved residues, quantifying the likelihood a residue will have a particular level of conservation given the conservation of the surrounding residues. The framework is expanded to assess the significance of groupings of conserved residues, a metric that forms the basis of SLiMPrints (short linear motif fingerprints), a de novo motif discovery tool. SLiMPrints identifies relatively overconstrained proximal groupings of residues within intrinsically disordered regions, indicative of putatively functional motifs. Finally, the human proteome is analysed to create a set of highly conserved putative motif instances, including a novel site on translation initiation factor eIF2A that may regulate translation through binding of eIF4E.
Figures
Figure 1.
(A–C) are sections of human Epsin 2 containing functionally important residues matching the regular expression for the AP2-binding motif DPW aligned against a selection of vertebrate Epsin 2 orthologues. Pairs of lower case letters denote amino acids flanking a region inserted compared with human Epsin 2 and the number specifies the length of the insertion. The alignment is coloured using the Clustal colouring scheme. (A and B) are known functional AP2-binding motifs and (C) is part of the N-Terminal ENTH domain. Lower panel: RLC scores for the section of the alignment (see ‘Materials and Methods’ section for a description of the RLC scoring scheme).
Figure 2.
Comparison of ‘non-ELM residues’ residues (white) against ‘ELM residues’ (grey) from the benchmarking data set from the ELM resource. (A) RLC value comparison, grey dashed line shows a Gaussian distribution (µ = 0, σ = 1). RLC values on _x_-axis are lower limits of bins of size 0.25. (B) _p_RLC value comparison, grey dashed line shows a uniform distribution. _p_RLC values on _x_-axis are lower limits of bins of size 0.05.
Figure 3.
Schema of the SLiMPrints method.
Figure 4.
Comparison of the _p_motif and _Sig_motif score distributions for ‘non ELM residues’ residues (grey) against ‘ELM residues’ (white). (A) _p_motif score distributions. _p_motif values on _x_-axis are lower limits of bins of size 0.1. (B) _Sig_motif score distributions. _Sig_motif values on _x_-axis are lower limits of bins of size 0.1.
Figure 5.
(A) ROC curve of CS (dashed and dotted), RLC (dashed) and _Sig_motif (solid) metrics. (B) Proportion of motifs returned at different _Sig_motif scores that are experimentally validated functional motifs from the ELM database.
Figure 6.
(A) Alignment of the 50 residues flanking the LxxFRxxWxxEL motif in FBXO9 orthologues showing the conservation across many diverse species, conserved residues are coloured by ClustalX colouring scheme. (B) Domain architecture of FBXO9 and FBXW8. Red diamond denotes position of LxxFRxxWxxEL motif. (C) Helical wheel representation of the LxxFRxxWxxEL motif.
Figure 7.
(A) Alignment of the 50 residues flanking the YxPPxLR motif in eIF2A orthologues showing the conservation across many diverse species, conserved residues are coloured by ClustalX colouring scheme. (B) Light grey boxes are domains involved in RNA metabolism, green domains are domains involved in translational regulation and grey domains have no obvious link to RNA processing. Red diamond denotes position of YxPPxLR motif. (C) Schematic of constructs used in assays with sequence variants shown. (D) Equal amounts of protein from S10 HeLa cell extracts were obtained after transfection with either no plasmid or wild-type or mutant forms of FLAG-tagged eIF2A and were subjected to SDS–PAGE and immunoblotting with the antibodies indicated (left hand panels). The extracts were then subjected to m7GTP Sepharose chromatography (right hand panel, lanes 2–5) to recover proteins associated with eIF4E. Untransfected cell extract was also incubated with control 4B Sepharose resin (lane 1). (E) Extracts from HeLa cells transfected as described in panel D were subjected to co-immunoprecipitation as described in ‘Materials and Methods’ section with AminoLink agarose resin coupled to FLAG-M2 antibody. Immunoblotting of proteins from the total cell extract (left hand panels) or eluted proteins (right hand panels) was carried out using the antibodies indicated.
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