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.

Keywords: biophysics; deubiquitylation (deubiquitination); structural biology; ubiquitin; ubiquitylation (ubiquitination).

FIGURE 1.

The structure of ubiquitin (Protein Data Bank: 1ubq). The main chain is colored red at the C terminus, and the lysine side chains are drawn and labeled. The side chains making up the two hydrophobic patches centered on Ile-44 and Ile-36 are drawn and colored orange and yellow, respectively. Two regions of ubiquitin have been shown to possess dynamics on the microsecond timescale. Amides displaying microsecond dynamics by spin relaxation experiments are drawn in blue (Ile-23, Glu-24, Asn-25, Glu-51, Asp-52, Gly-53, Thr-55, and Val-70). Amides in the β1-β2 loop that are conformationally mobile in the RDC ensemble are drawn in purple (Thr-7 through Leu-11).

FIGURE 2.

Differentially linked ubiquitin chains adopt diverse conformations. Monoubiquitin (top) is colored as in Fig. 1, with the Ile-44 and Ile-36 hydrophobic interaction patches in orange and yellow and the C terminus in red. Lys-6, Lys-11, and Lys-48 adopt compact conformations, with the interaction patches differentially exposed in each arrangement. Conversely, Lys-63-linked and linear chains are quite extended. Lys-11 and Lys-48 chains can sample other orientations about their preferred compact conformation, but little is known about the dynamics of Lys-6 chains. Because Lys-63 and linear chains are unconstrained, they are free to adopt a wide range of arrangements.

FIGURE 3.

Autoinhibited E3 ligases require conformational changes for activation and possess residual internal dynamics that may couple to function. a, several crystal structures have captured Parkin in an autoinhibited conformation with both its active site cysteine and its E2-binding site occluded by a dense network of intradomain interactions (71–73). The dynamics of the Ubl domain are implied by the fact that its Ile-44 patch must undock from the RING1 domain to accommodate known binding partners and the observation that the Ubl is dispensable for folding of the rest of the protein. The molecular details of Parkin activation are still being investigated (9, 77–79), but the E2-binding site on the RING1 domain and the active site cysteine on RING2 must be accessible for Parkin ligase activity. b, cIAP1 adopts an autoinhibited conformation that is disrupted upon association with SMAC or SMAC mimetics. Significant residual motions in the autoinhibited state have been experimentally identified, and these motions seem to be associated with a fast transition to an active ligase dimer upon the binding of SMAC mimetics (81, 84).