Using Protein Motion to Read, Write, and Erase Ubiquitin Signals

Abstract

Eukaryotes use a tiny protein called ubiquitin to send a variety of signals, most often by post-translationally attaching ubiquitins to substrate proteins and to each other, thereby forming polyubiquitin chains. A combination of biophysical, biochemical, and biological studies has shown that complex macromolecular dynamics are central to many aspects of ubiquitin signaling. This review focuses on how equilibrium fluctuations and coordinated motions of ubiquitin itself, the ubiquitin conjugation machinery, and deubiquitinating enzymes enable activity and regulation on many levels, with implications for how such a tiny protein can send so many signals.Ubiquitin was first identified as a small, highly conserved, and heat-stable protein ubiquitously expressed in all eukaryotes and was later shown to be a central regulator of protein homeostasis by directing substrates to the proteasome (1). Since this seminal work in the late 1970s and early 1980s, the covalent modification of substrates with ubiquitin and ubiquitin chains has been shown to control myriad cellular processes (2) and has also been implicated in the pathology and potentially the treatment of numerous diseases (3).Ubiquitin is typically attached to substrate proteins via an isopeptide bond between the flexible C terminus of ubiquitin and the ⑀-amino group of a substrate lysine, although substrate proteins can also be ubiquitinated at their N terminus. The initial modification can be differentiated by conjugation of additional ubiquitin molecules at any of ubiquitin's seven lysine residues or its own N terminus, resulting in the formation of ubiquitin chains. These different chain linkages encode different signals, and tens of thousands of protein isoforms are ubiquitinated in human cells, implying a massive potential regulatory impact for the cell (4). Lys-48 polyubiquitination is the canonical ubiquitin signal marking substrates for degradation by the proteasome (5), although Lys-11 chains have also been shown to encode degradative signals, particularly in the regulation of mitosis (6). Lys-63 and linear chains are involved in driving substrates to specific signaling complexes, including those involved in NF-B signaling (7), whereas Lys-6 chains have been implicated in autophagy (8,9). Although all possible linkages have been detected in cells (10, 11), the functions of Lys-27, Lys-29, and Lys-33 chains are still emerging. The possibility of branched chains and chains of mixed linkages provides further complexity. Because the literature on ubiquitination is vast, we will direct the reader to more exhaustive reviews covering other aspects of ubiquitin signaling throughout this minireview.Ubiquitination of substrates is controlled by the action of three families of enzymes: the ubiquitin activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). Ubiquitin modifications are edited or removed from substrates by deubiquitinating enzymes (DUBs).3 In humans, there are two E1s, 30 -40 E2s, over 600 E3s, and over 100 DUBs, resul...

Conformational Dynamics of Deubiquitinase A and Functional Implications

“…Structural and biochemical studies of other DUBs also suggest the functional relevance of conformational plasticity. Evidence suggests that the structures of many DUBs in the apo state are heterogeneous and can be modulated by binding of accessary domains, , partner proteins, − substrates, , and small-molecule inhibitors, thereby controlling enzyme activity and/or substrate specificity. Recent work on USP7 has also shown that point mutations can shift this DUB from the inactive to an active state, supporting the notion that DUBs can be activated through subtle changes in conformational ensembles .…”

An invisible ubiquitin conformation is required for efficient phosphorylation by PINK 1

“…NMR studies in particular have shown that despite its compact fold and high intrinsic stability, Ub is dynamic and contains several regions of local conformational flexibility (Lange et al, 2008). These include a mobile fourresidue C-terminal tail, as well as a flexible b-hairpin structure, the b1/b2-loop, that alters the interaction profile of Ub (Lange et al, 2008;Hospenthal et al, 2013;Phillips & Corn, 2015). Importantly, we previously discovered that Ser65 phosphorylation resulted in a further, dramatic conformational change in Ub (Wauer et al, 2015a).…”

An ‘invisible’ ubiquitin conformation is required for efficient phosphorylation by PINK1

“…NMR studies in particular have shown that despite its compact fold and high intrinsic stability, Ub is very dynamic and contains several regions of local conformational flexibility (Lange et al , 2008). These include a mobile four-residue C-terminal tail, as well as a flexible β hairpin structure, the β 1/ β 2-loop, that alters the interaction profile of Ub (Lange et al , 2008; Hospenthal et al , 2013; Phillips & Corn, 2015). Importantly, we previously discovered that Ser65-phosphorylation resulted in a further, dramatic conformational change in Ub (Wauer et al , 2015b).…”