Macromolecular juggling by ubiquitylation enzymes

Abstract

The posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins is accomplished by the sequential action of E1, E2, and E3 enzymes. Members of the E1 and E3 enzyme families can undergo particularly large conformational changes during their catalytic cycles, involving the remodeling of domain interfaces. This enables the efficient, directed and regulated handover of ubiquitin from one carrier to the next one. We review some of these conformational transformations, as revealed by crystallographic studies.

Figure 1

Ubiquitylation is a multistep reaction. (a) E1 enzymes use ATP to activate the carboxyl terminus of ubiquitin (Ubi) as a high-energy anhydride (Ubi-AMP). The E1 active site cysteine then attacks the adenylated ubiquitin to form a thioester intermediate. Subsequently, the active site cysteine of the E2 receives ubiquitin via trans-thioesterification. (b) E3 enzymes catalyze the formation of an isopeptide bond between the ubiquitin carboxyl terminus and a primary amino group of an acceptor. The acceptor can be a target protein (mono-ubiquitylation/ubiquitin chain initiation) or another ubiquitin molecule (ubiquitin chain elongation). Catalysis by HECT- and RBR-type E3 enzymes proceeds through an intermediate, in which the ubiquitin carboxyl terminus is thioester-linked to a cysteine residue at the active site of the E3, followed by aminolysis of the thioester. In contrast, RING-type E3s catalyze direct transfer of ubiquitin from the E2 active site cysteine to amino groups on the acceptor.

Figure 2

Conformational rearrangements in E1 enzymes. Cartoon representations of distinct states in the catalytic cycle of canonical E1 enzymes. (a) The adenylation state based on the crystal structure of NAE1-UBA3 in complex with NEDD8 and ATP/Mg²⁺ [PDB: 1R4N] [32]. The carboxy-terminal tail of the Ubl is in the adenylation site of the active Rossmann-type subunit of the E1, ready to nucleophilically attack the α-phosphate of the ATP to form the Ubl-AMP intermediate. The catalytic cysteine residue in the E1 cysteine domain is part of an α-helix and is removed from the adenylation site, giving rise to an open conformation of the cysteine domain. (b) The thioesterification state as seen in a crystal structure of SAE1-UBA2 and SUMO covalently coupled to an AMP analogue that mimics the tetrahedral intermediate generated during thioesterification [PDB: 3KYD] [8]. Mediated by large conformational changes in the crossover and re-entry loops, the cysteine domain is rotated with respect to the Rossmann-type subunits. The helix containing the active site cysteine seen in (a) has melted. In this closed conformation of the cysteine domain, the catalytic cysteine nucleophile is in position to attack the adenylated carboxyl terminus of the Ubl. The positive dipole of helix H2 in the active Rossmann-type subunit (colored purple) is thought to favor this reaction [8]. (c) The trans-thioesterification state as represented by a crystal structure of NAE1-UBA3 thioester-linked to NEDD8 and in complex with an additional NEDD8 molecule, an E2 enzyme (Ubc12) and ATP/Mg²⁺[35]. The cysteine domain of the E1 adopts an open orientation similar to the adenylation state (a), but now holds the carboxyl terminus of the thioester-linked Ubl close to the E2 active site (a Cys-to-Ala mutant of the E2 was used in this study (see text)). The ubiquitin-fold domain has swung away from its position in the previous states (a,b) to accommodate the E2 and contributes to E2 binding. In (a,c) domains found in NAE1-UBA3 but not in SAE1-UBA2 were omitted for clarity. To see a rendition of a dynamic transition between the structures shown in the lower panels of (a-c), see Additional file 1. As noted in the movie legend, the details of the trajectory linking individual structures is not realistic and is simply meant to illustrate the nature of the conformational changes rather than identify the nature of the transition pathway.

Figure 3

Swinging domains in HECT E3 enzymes. Cartoon representations of crystal structures of various HECT domains. (a) Open, ‘L’-shaped conformation of E6AP (E3) in complex with UbcH7 (E2) [PDB: 1C4Z] [62], (b) closed, ‘T’-shaped conformation of WWP1/AIP [PDB: 1ND7] [63], and (c) trans-thioesterification complex of NEDD4L with a ubiquitin-E2 (UbcH5B) conjugate [PDB:3JVZ] [64]. In (c) the E2 active site cysteine was mutated to serine (colored yellow in our representation), resulting in an oxy-ester linkage with ubiquitin in lieu of the native thioester. (d) Distinct classes of C-lobe orientations based on the crystal structures of various HECT domains (WWP1/AIP [PDB: 1ND7], Itch [PDB: 3TUG], HUWE1 [PDB: 3G1N, 3H1D], NEDD4L [PDB: 2ONI, 3JVZ], E6AP [PDB: 1C4Z], Rsp5 [PDB: 3OLM], Smurf2 [PDB: 1ZVD], NEDD [PDB: 2XBB]). A second unique C-lobe orientation observed for NEDD [PDB: 2XBF] could not be displayed for clarity. In our representation the HECT N-lobes are superimposed and only one of them is displayed. Binding partners, such as E2 enzymes or ubiquitin, found in some of the structures are not displayed.

Figure 4

Regulatory rearrangements in Cbl proteins. (a) ‘Closed’ conformation of Cbl based on the crystal structure of the apo c-Cbl amino-terminal region, comprising the tyrosine kinase binding module, the helical linker region, and the RING domain [PDB: 2Y1M] [29]. The regulatory tyrosine, Y371, located in the helical linker region, is buried in a hydrophobic core formed by the SH2 domain and the four-helix bundle in the tyrosine kinase binding module. (b) ‘Partially open’ conformation of Cbl based on the co-crystal structure of c-Cbl amino-terminal region with a ZAP70-derived phosphopeptide and the E2 enzyme UbcH7 [PDB: 1FBV] [91]. Phosphopeptide binding induces a shift in the SH2 domain that perturbs the interface between the helical linker and the tyrosine kinase binding module, probably favoring dissociation of the RING domain from the tyrosine kinase binding module and thus increasing the accessibility of the E2 binding surface. (c) ‘Open’ conformation of Cbl based on the co-crystal structure of phosphorylated c-Cbl bound to a ZAP7-derived phosphopeptide and UbcH5B [PDB: 4A4C] [29]. The phosphorylated regulatory tyrosine, Tyr371, interacts with residues in the E2 binding surface of the RING domain. The RING domain is situated on the opposite side of the tyrosine kinase binding module compared to (b).