A context-dependent and disordered ubiquitin-binding motif

Abstract

Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds ubiquitin to translate and effectuate this posttranslational signal. Recent computational studies have suggested that protein regions can recognize ubiquitin via a process of folding upon binding. Using peptide binding arrays, bioinformatics, and NMR spectroscopy, we have uncovered a disordered ubiquitin-binding motif that likely remains disordered when bound and thus expands the palette of ubiquitin-binding proteins. We term this motif Disordered Ubiquitin-Binding Motif (DisUBM) and find it to be present in many proteins with known or predicted functions in degradation and transcription. We decompose the determinants of the motif showing it to rely on features of aromatic and negatively charged residues, and less so on distinct sequence positions in line with its disordered nature. We show that the affinity of the motif is low and moldable by the surrounding disordered chain, allowing for an enhanced interaction surface with ubiquitin, whereby the affinity increases ~ tenfold. Further affinity optimization using peptide arrays pushed the affinity into the low micromolar range, but compromised context dependence. Finally, we find that DisUBMs can emerge from unbiased screening of randomized peptide libraries, featuring in de novo cyclic peptides selected to bind ubiquitin chains. We suggest that naturally occurring DisUBMs can recognize ubiquitin as a posttranslational signal to act as affinity enhancers in IDPs that bind to folded and ubiquitylated binding partners.

Deciphering the Alphabet of Disorder—Glu and Asp Act Differently on Local but Not Global Properties

“…The broadening or loss of signals makes it difficult to quantify binding affinity; thus, in a recent study, we titrated 15 N-ubiquitin with unlabeled Dss1 to circumvent the loss of signal intensity to quantify the affinity of WT Dss1 for mono-ubiquitin giving a K d of 380 μM [ 24 ]. The titration of the variants with ubiquitin into 40 molar excess showed smaller CSPs than WT Dss1 ( Figure 4 B and Figure S4 ), suggesting weaker affinities and a smaller population of the bound state.…”

“…In the proteasome, Dss1 functions as a ubiquitin-receptor, binding polyubiquitylated substrates destined for degradation. Thus, while Dss1 binds mono-ubiquitin, it can also bind chains of ubiquitin, exploiting two disordered ubiquitin-binding motifs [ 24 ] (UBS) I from D38–D49 and UBS II from D16–N25, with UBS I has the strongest affinity for ubiquitin [ 23 ]. A transiently formed C-terminal helix in Dss1 interacts dynamically with UBS I, and this fold-back structure may function to shield the binding site and thereby regulate binding partner interaction [ 19 ].…”

Deciphering the alphabet of disorder — Glu and Asp act differently on local but not global properties

“…Broadening or loss of signals makes it difficult to quantify binding affinity; thus in a recent study we titrated 15 N-ubiquitin with unlabelled Dss1 to circumvent the loss of signal intensity to quantify the affinity of WT Dss1 for mono-ubiquitin to give a K d of 380 μM [70] Titration of the variants with ubiquitin into 40 molar excess showed smaller CSPs than WT Dss1 (Figure 4B and Figure S4) suggesting weaker afiinities and lower population of the bound state. However, since ubiquitin is known to form dimers at mM concentrations [71], we were unable to reach saturation.…”

“…The C-terminal region adapts an α-helical conformation when bound, which also exists transiently when free in solution [21]. Dss1 binds mono ubiquitin at two binding sites exploiting a disordered ubiquitin binding motif [22] (UBS) I from D38-D49 and UBS II from D16-N25, with UBS I having the strongest affinity for ubiquitin [23]. The transient C-terminal helix interacts dynamically with UBS I, and this fold-back mechanism may function to shield the binding site and thereby regulate binding partner interaction [17].…”

“…In the proteasome, Dss1 may function as a ubiquitin receptor binding polyubiquitylated substrates destined for degradation. Thus, while Dss1 binds mono-ubiquitin, it can also bind chains of ubiquitin, exploiting two disordered ubiquitin binding motif [22] (UBS) I from D38-D49 and UBS II from D16-N25, with UBS I having the strongest affinity for ubiquitin [23]. A transiently formed C-terminal helix in Dss1 interacts dynamically with UBS I, and this fold-back mechanism may function to shield the binding site and thereby regulate binding partner interaction [17].…”