Bringing order to protein disorder through comparative genomics and genetic interactions

Abstract

Background: Intrinsically disordered regions are widespread, especially in proteomes of higher eukaryotes. Recently, protein disorder has been associated with a wide variety of cellular processes and has been implicated in several human diseases. Despite its apparent functional importance, the sheer range of different roles played by protein disorder often makes its exact contribution difficult to interpret.

Results: We attempt to better understand the different roles of disorder using a novel analysis that leverages both comparative genomics and genetic interactions. Strikingly, we find that disorder can be partitioned into three biologically distinct phenomena: regions where disorder is conserved but with quickly evolving amino acid sequences (flexible disorder); regions of conserved disorder with also highly conserved amino acid sequences (constrained disorder); and, lastly, non-conserved disorder. Flexible disorder bears many of the characteristics commonly attributed to disorder and is associated with signaling pathways and multi-functionality. Conversely, constrained disorder has markedly different functional attributes and is involved in RNA binding and protein chaperones. Finally, non-conserved disorder lacks clear functional hallmarks based on our analysis.

Conclusions: Our new perspective on protein disorder clarifies a variety of previous results by putting them into a systematic framework. Moreover, the clear and distinct functional association of flexible and constrained disorder will allow for new approaches and more specific algorithms for disorder detection in a functional context. Finally, in flexible disordered regions, we demonstrate clear evolutionary selection of protein disorder with little selection on primary structure, which has important implications for sequence-based studies of protein structure and evolution.

Figure 1

Genetic interactions distinguish different roles of disorder. (a) Percentage of disordered residues of yeast proteins by their number of GIs. (b) Multi-functionality (see Materials and methods) for disordered and structured GI hubs and non-hubs. Hubs are genes in the top 90th percentile (above 90 interactions) of GIs while non-hubs are in the bottom 50th percentile (below 15 interactions). (c) Evolutionary constraint on sequence (dN/dS ratio) on hubs and non-hubs. In both cases disordered proteins have a significantly higher dN/dS than structured proteins. (d) Evolutionary constraint measured by the presence of orthologs in other yeast species (phylogenetic persistence). While disordered non-hubs are less conserved than structured non-hubs, the disordered hubs are as conserved as structured hubs. P-values were computed with a Wilcoxon test, and error bars represent boot-strapped 95% confidence intervals.

Figure 2

Two forms of conservation on disorder. Schematic of computing disorder conservation and amino acid (AA) sequence conservation. After alignment, the percentage of sequences in which a residue is disordered is computed. Similarly, we compute the percentage of sequences in which the amino acid itself is conserved. A residue is considered to be conserved disorder if the property of disorder is conserved in ≥ 50% of species and sequentially conserved if the amino acid is conserved in ≥ 50% of species. Disordered residues in which both sequence and disorder are conserved are referred to as constrained disorder. Disordered residues in which disorder is conserved but not the amino acid sequence are referred to as flexible disorder. Residues which are disordered in S. Cerevisiae but not cases of conserved disorder are referred to as non-conserved disorder.

Figure 3

Densities of disorder- and amino acid-conserved residues by their scores. Densities of disorder and amino acid conservation scores across all alignments of approximately 5,000 orthologous groups from 23 yeast species. (a) Histogram of the amino acid (AA) conservation scores. (b) Histogram of disorder conservation scores. (c) Two-dimensional histogram of both amino acid and disorder conservation scores.

Figure 4

Properties associated with types of disorder. Correlation coefficients of different genomic features with percent constrained disorder, percent flexible disorder and percent non-conserved disorder. Error bars represent 95% confidence intervals.

Figure 5

Properties associated with types of disorder. (a) Heatmap of enrichment (density over background) of phosphosites in terms of disorder and amino acid conservation. (b) Partial correlation of phosphosite density and disorder conservation with respect to amino acid conservation (see Materials and methods). (c) Partial correlation of phosphosite density and conserved amino acid sequence with respect to disorder conservation.

Figure 6

Singlish and multi-interface hubs have different proportions of flexible and constrained disorder. The mean proportion of flexible disorder and constrained disorder in singlish-interface and multi-interface protein interaction hubs. While both have a similar level of constrained disorder, singlish hubs are heavily enriched for flexible disorder. Error bars represent 95% confidence intervals.

Figure 7

Disorder splits into three distinct phenomena. Functional enrichment maps of proteins enriched in flexible disorder versus constrained disorder. The area of each rectangle is proportional to the representation of that type of disorder in the alignments. Related GO terms are grouped based on gene overlap (see Materials and methods; Figures S20, S21 and S22 in Additional file 1).