RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination - PubMed (original) (raw)
Review
RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination
Meredith B Metzger et al. Biochim Biophys Acta. 2014 Jan.
Abstract
RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Keywords: Catalysis; Protein degradation; RING finger; U-box; Ubiquitin ligase (E3); Ubiquitin-conjugating enzyme (E2).
Published by Elsevier B.V.
Figures
Figure 1. Ubiquitination
Protein ubiquitination requires the sequential action of three classes of enzymes. First, ubiquitin (Ub) is activated in an ATP-dependent manner by ubiquitin-activating enzyme (E1). In this step, the C-terminus of ubiquitin is linked by a thioester bond to the active site Cys of E1 (E1~Ub). Ubiquitin is then transthiolated to the active site Cys of a ubiquitin-conjugating enzyme (E2), generating an E2~Ub thioester. Ubiquitin protein ligases (E3) interact with both E2~Ub and the substrate to which ubiquitin is to be transferred, thus providing much of the specificity in the ubiquitin system. HECT-type E3s function as covalent intermediates in the ubiquitination pathway as ubiquitin is first transferred to the active site Cys of E3 via a transthiolation before conjugation to substrate. RING-type E3s instead mediate the transfer of ubiquitin directly from E2~Ub to substrate. Ubiquitination generally occurs on primary amines (Lys, and, less frequently, a free N-terminus) as a consequence of nucleophillic attack on the E2~Ub linkage, resulting in stable isopeptide (or peptide) linkages with the C-terminus of ubiquitin. Monoubiquitination plays diverse roles in processes such as DNA repair, protein trafficking, and transcription [159], and recent findings have demonstrated that multi-monoubiquitination can also target proteins for proteasomal degradation [160, 161]. Alternatively, ubiquitin can be transferred to one of the seven Lys (K) or the N-terminal Met of ubiquitin molecules [162] (PDB 1UBQ) that are already substrate-linked, generating multi- or poly-ubiquitin chains. To a large extent, the nature of the ubiquitin linkages specifies the fate and function of the modified protein. For example, chains of four or more ubiquitins linked through K48 efficiently target proteins for proteasomal degradation [163]. Similarly, K11-linked chains are integral to proteasomal targeting of anaphase-promoting complex/cyclosome (APC/C) substrates [164] and chains linked through other lysines may also play a role in proteasomal targeting [165, 166]. K63-linked and linear ubiquitin chains are implicated in non-proteasomal aspects of NF-κB signaling [167]. K63-linked chains and mono-ubiquitination are also implicated in DNA repair and the targeting of cell surface and endocytic proteins for lysosomal degradation [168, 169].
Figure 2. RING domains coordinate Zn2+ in a crossbrace arrangement that serves as a platform for E2 binding
A) Representation of the crystal structure of the TRAF6 RING domain (blue) bound to the E2, Ubc13 (green) [89] (PDB 3HCT) highlights a stereotypical RING:E2 interaction. The catalytic Cys of Ubc13 is highlighted in yellow, while its RING domain-interacting regions are in purple. Yellow TRAF6 RING residues with sidechains shown are those that coordinate Zn2+ (C3HC3D), forming the RING crossbrace structure modeled in B). The two loops (Zn I, Zn II) and the intervening central α-helix formed by this structure together serve as a conserved platform for E2 binding. B, C) Model of the interleaved RING crossbrace structure (B) and consensus sequence (C). The eight Zn2+-coordinating residues are shown in yellow and X is any amino acid.
Figure 3. Architecture of RING-type E3s
A) Model of a monomeric RING-type E3, where its RING domain would mediate binding to E2 thioester-linked to ubiquitin. Binding to substrate occurs generally through regions of the E3 other than the RING domain. B) Model of a multi-subunit RING E3 of the Cullin RING Ligase (CRL) superfamily, such as the well-studied SCF (CRL1) family, shown here. SCF consists of a cullin protein (Cul1) a small RING finger protein (Rbx1), and an adaptor protein (Skp1) that binds interchangeable substrate recognition elements (F-box proteins). The ubiquitin-like molecule, Nedd8, is reversibly conjugated to cullins and associated with activation of CRLs. C) Schematic of dimeric RING E3s, such as cIAP, RNF4, BIRC7, IDOL, Mdm2-MdmX, that dimerize through their RING domains and interleaved C-terminal tails. D) Ribbon diagram illustrating the homodimeric RING E3, BIRC7 [13] (PDB 4AUQ) as a representative of the class of dimers schematized in C. The E2-interacting residues of one RING domain are highlighted in red. E) Model of dimeric RING E3s, such as BRCA1-BARD1 and RING1B-Bmi1, where α-helices both N- and C-terminal to the RING facilitate dimerization. In the case of BRCA1-BARD1 (illustrated), this occurs through a four α-helix bundle (helices above RINGs in F). F) Ribbon diagram illustrating the heterodimeric RING dimer of BRCA1-BARD1 [19] (PDB 1JM7) modeled in E). The E2-interacting residues of the RING domain of BRCA1 are highlighted in red.
Figure 4. Activation of E2~Ub conjugates by RING-type domains
A) Schematic of ubiquitin thioester linked to E2 sampling ‘open’ and ‘closed’ confirmations in the absence of a RING domain (left). RING binding to E2~Ub promotes increased occupancy of closed confirmations needed for ubiquitin transfer (right). B) Ribbon diagram of BIRC7-BIRC7:UbcH5B~Ub (PDB 4AUQ) as an example of a RING-type E3:E2~Ub ternary complex. E3, E2, and ubiquitin are blue, green, and red, respectively. The catalytic Cys of UbcH5B is labeled. Gray circles are the zincs coordinated by the BIRC7 RINGs. C) The ribbon diagram shown in B, with the ubiquitin molecule removed and the residues on E2 or the RING domains that would contact ubiquitin in red. The 310 helix, helix 2, and catalytic Cys of UbcH5B are labeled. D) A closer view of the E2 active site and ubiquitin C-terminus from the BIRC7-BIRC7:UbcH5B~Ub structure shown in B and C. The UbcH5B helix 2 (green) is positioned directly above Arg72. Residues from BIRC7, UbcH5B, and ubiquitin are underlined in blue, green, and red respectively. The hydrogen bonding network created by BIRC7 Arg286 (blue) and the E2 backbone of Gln92, the ubiquitin backbone of Arg72, and the side chain of ubiquitin Gln40 is shown. Residues colored in purple correspond to those that display NMR spectral effects specifically arising from the E3:E2 hydrogen bond. The ‘up’ conformation of the Asp87 side chain seen in this structure is shown in purple and red, the ‘down’ conformation, frequently seen in structures in the absence of covalently-bound ubiquitin is shown coming off the backbone directly below in semi-transparent cyan and orange (taken from PDB 3UGB [170]). Contacts made by the ‘up’ Asp87 conformation to Arg74 (red) of ubiquitin are shown. The side chains of UbcH5B Gln92, Ub Arg72, and Ub Arg74 are not shown for clarity.
Figure 5. E2 binding domains other than the RING modulate ubiquitination by binding to the ‘backside’ of E2s
A–F) Structures of A) ubiquitin (red, PDB 2FUH [102]); B) SUMO (orange, PDB 2UYZ [151]); or the non-RING E2 binding domains (blue) found in C) Rad18 (R6BD, PDB 2YBF [84]); D) gp78 (G2BR, PDB 3H8K [83]); E) Cue1p (U7BR, PDB 4JQU [141]); or F) Pex22p (Pex22pS, PDB 2Y9M [148]) binding to the ‘backside’ of their respective E2s (labeled and shown in green). The catalytic cysteine of each E2 is highlighted in yellow. The thioester-linked ubiquitin interaction surface is colored in magenta in panel A.
References
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