Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways

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Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.

Introduction

Post-translational protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers regulates a variety of cellular processes, including protein degradation, signaling, sorting, localization, activation and repression [1]. There are up to 14 distinct Ub/Ubl families, and while diverse at the primary sequence level, most exhibit similar three-dimensional folds. In instances where Ub/Ubls impart their activities through covalent attachment to substrates, Ub/Ubl pathways utilize analogous enzymes to facilitate covalent isopeptide bond formation between the Ub/Ubl C-terminus and substrate lysine (Figure 1a) [2].

Ub/Ubl modifiers are translated as precursors, and proteases must process Ub/Ubls to expose a C-terminal Gly–Gly motif which is then adenylated by E1. The adenylate is attacked by a conserved E1 cysteine to form a thioester bond, releasing AMP. The E1∼Ub/Ubl (where ‘∼’ denotes a thioester bond) recruits an E2 conjugating enzyme whereupon the Ub/Ubl thioester is transferred from the E1 cysteine to a conserved E2 cysteine. E3 ligases facilitate Ub/Ubl transfer by coordinating both E2∼Ub/Ubl and substrate to ensure specificity and to enhance conjugation. Once conjugated, the ‘Ub/Ubl signal’ can be recognized by proteins via Ub/Ubl binding domains. To terminate the signal, Ub/Ubl proteases deconjugate Ub/Ubl from the substrate.

We will present recent structural insights into each step of Ub/Ubl modification – including those relevant to conjugation, Ub/Ubl-substrate recognition, and deconjugation. Although each step in the Ub/Ubl pathway could sustain a comprehensive review in Current Opinion in Structural Biology, we will limit our discussion to selected structural studies involving ubiquitin, Nedd8, or SUMO to illuminate mechanisms pertinent to Ub/Ubl modification.

Section snippets

E1 activating enzymes

Structures are available for two heterodimeric E1s that facilitate Ub/Ubl activation and transfer to E2 conjugating proteins, including the Nedd8 E1 (APPBP1/Uba3) and the SUMO E1 (Aos1/Uba2 or SAE1/SAE2) [3, 4, 5]. These structures revealed three essential E1 domains, the Ub/Ubl adenylation domain, the Cys domain which contains the cysteine required for thioester transfer, and a ubiquitin-fold domain (UFD) responsible for E2 recruitment (Figure 1b and c).

Structures of APPBP1/Uba3-Nedd8-ATP and

E2 conjugating enzymes

E2 conjugating enzymes accept Ub/Ubls from E1 through thioester bond exchange, resulting in a ‘charged’ E2∼Ub/Ubl that can transfer the ‘donor’ Ub/Ubl to a target lysine to form an isopeptide bond between the Ub/Ubl C-terminus and the Nζ atom of the target lysine. Isopeptide bond formation requires attack at the Ub/Ubl-thioester carbonyl carbon by the nucleophilic acceptor lysine. E2 catalytic cores share a conserved structure, but apart from the E2 cysteine, few discernable ‘catalytic’

E3 ligases

E3 Ub/Ubl ligases promote conjugation by bringing charged E2∼Ub/Ubls and substrates into close proximity. E3 ligases fall into two broad categories—the RING/U-box and HECT domain families. The RING/U-box E3s utilize a RING/U-box domain to recruit the E2∼Ub/Ubl while the HECT domains recruit E2∼Ub/Ubl to facilitate formation of an E3∼Ub/Ubl thioester with a conserved HECT cysteine prior to substrate conjugation. Structural studies have been focused on resolving E3-substrate interactions and the

Ub/Ubl recognition

There are over 16 classes of Ub recognition domains [33, 34]. While the majority is composed of α-helices, a few are known to contain zinc-binding motifs. Most of these interact with a hydrophobic patch on the ubiquitin surface surrounding Ile44, although they do so using different strategies. For example, UIM domains recognize the hydrophobic patch using a single α-helix (Figure 4b, [35]), while CUE domains consist of a three helix bundle which interacts with the hydrophobic patch through

Ub/Ubl proteases

Most Ub/Ubl proteases fall within the papain-like protease family, and catalysis is achieved via a catalytic-triad composed of side chains from a nucleophilic cysteine, adjacent histidine, and stabilizing asparagine/aspartic acid residue. Recent structures for several different classes of Ub/Ubl deconjugating enzymes have revealed mechanisms pertinent to protease regulation and Ub/Ubl substrate recognition [41].

The structures of two UBP Ub proteases illustrate discreet mechanisms whereby Ub

Conclusions and future directions

A remarkable explosion of new structures in the Ub/Ubl field has occurred since the last article appeared in Current Opinion in Structure Biology in 2002 [49]. While many pertinent issues have been resolved through structure determination of protein complexes involved in Ub/Ubl conjugation and deconjugation, much work remains to be done. We have attempted to construct models that illustrate mechanisms pertinent to Ub/Ubl pathways by cobbling together results from studies in ubiquitin, Nedd8, or

References and recommended reading

  • • of special interest

  • •• of outstanding interest

Acknowledgements

A.D.C. and C.D.L. are supported in part by a grant from the National Institutes of Health (GM65872). A.D.C. acknowledges support from the NIH (GM075695).

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