Mining the TRAF6/p62 interactome for a selective ubiquitination motif

Abstract

A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

Figure 1

Model illustrating the interaction between the ligase and scaffold in directing substrate ubiquitination. A: Schematic representation of the means by which the E3 ligase, TRAF6, interacts with the scaffold, p62, and selects a specific Lysine for ubiquitination. B: The sequence of the consensus motif identified in TRAF6/p62 substrates.

Figure 2

Motif match search results. A: Percentage distributions for positive matches at 1, 2, 3, 4, 5, 6, and 7 of the variable sites within the 10 amino acid long putative motif. The number of proteins in each dataset was: Experimental = 155, Negative = 54, Randomized = 999. (insert). Percentages for a subset of positive hits (5, 6, 7) hypothesized to be biological meaningful. B: Actual counts of the number of matches observed in each dataset for each level of positive match.

Figure 3

Probability – Probability (P/P) plots illustrating the relationships between values predicted under Discrete Uniform Distribution models and empirical motif match results. A: P/P plot for the experimental dataset match distribution. B: P/P plot for the negative dataset match distribution. C: P/P plot for the randomized dataset match distribution

Figure 4

Sequence logos illustrating the occurrence of amino acids at each site within the putative motif. A: Frequency distribution of amino acids surrounding Lysines (K) with positive hits at seven variable positions in a ten amino acid long consensus motif in the experimental dataset. B: The distribution at six variable positions in a ten amino acid long consensus motif in the experimental dataset. C: The distribution at six variable positions in a ten amino acid long consensus motif in the negative dataset. K (red); AFGILMV (blue); CQSY (green); DHT (orange).

Figure 5

Structural context of predicted TRAF6/p62 ubiquitination sites within 30 proteins containing perfect motif matches. A: Distribution of perfect match motif sites based on secondary structure for the experimental and negative datasets. B: Percentage distribution of predicted sites in disordered protein regions and domain structures. C: Relative proportions of perfect match sites found in three protein regions, N-terminus, Middle and C-terminus. D: Identification of perfect match Lysines as likely occupying exposed or buried protein regions as determined by solvent accessibility predictions.

Figure 6

Sub-cellular localization of TRAF6/p62 substrates. Comparisons presented for each database showing the total proportion of proteins in each category versus the proportion predicted among the perfect and near perfect motif match proteins.