Structural and functional insights to ubiquitin-like protein conjugation

Abstract

Attachment of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins regulates numerous cellular processes including transcription, the cell cycle, stress responses, DNA repair, apoptosis, immune responses, and autophagy, to name a few. The mechanistically parallel but functionally distinct conjugation pathways typically require the concerted activities of three types of protein: E1 Ubl-activating enzymes, E2 Ubl carrier proteins, and E3 Ubl ligases. E1 enzymes initiate pathway specificity for each cascade by recognizing and activating cognate Ubls, followed by catalyzing Ubl transfer to cognate E2 protein(s). Under certain circumstances, the E2 Ubl complex can direct ligation to the target protein, but most often requires the cooperative activity of E3 ligases. Reviewed here are recent structural and functional studies that improve our mechanistic understanding of E1-, E2-, and E3-mediated Ubl conjugation.

Keywords: E1; E2; E3; ligase; ubiquitin; ubiquitin-like protein.

Figure 1

E1 ubiquitin-like protein (Ubl) activating enzymes. (a) Ubl conjugation cascade. (b) Small ubiquitin-like modifier protein (SUMO)-adenylate bound to SUMO E1 in the open conformation (PDB ID: 3KYC). Two helices within the CYS domain have been colored orange and cyan to illustrate the CYS domain movement relative to panel c. (c) SUMO-adenylate-SUMO E1 tetrahedral intermediate complex in the closed conformation (PDB ID: 3KYD). Two helices within the CYS domain have been colored orange and cyan to illustrate the CYS domain movement relative to panel b. (d) Nedd8 E1 with E2_Ubc12, Nedd8 adenylate and Nedd8 thioester bound (PDB ID: 2NVU). (e) E1–E2_Ubc4 cross-linked complex with ubiquitin (Ub) bound to the adenylation site (PDB ID: 4II2). (f) Model of E1_Atg7–E2_Atg3 cross-linked structure with Ubl_Atg8 bound, generated by alignment of molecules with the following PDB IDs: 3VH2, 3VH4, and 4GSL. E1 and E2 active-site cysteines are shown as yellow spheres, and magnesium ions are shown as red spheres. Molecular images generated with PyMOL (see Reference 92). Abbreviations: AAD, active adenylation domain; CTD, C-terminal domain; CYS, second catalytic cysteine half-domain (with active-site cysteines); ECTD, extreme C-terminal domain; FCCH, first catalytic cysteine half-domain; IAD, inactive adenylation domain; NTD, N-terminal domain; PDB, Protein Data Bank; UFD, ubiquitin fold domain.

Figure 2

Dynamics of E2~Ubl regulation. (a) E2 alignment of E2–Ubl thioester mimetic structures. For clarity, only E2_Ubc1 (from PDB ID: 1FXT) is shown; the varied Ub positions are shown for the indicated PDB IDs. (b) E2 alignment of E3-bound E2–Ubl thioester mimetics (E2_Ubc1–Ub from 1FXT is included for reference). E3 species have been removed for clarity. (c) Alignment of backside-binding E2 regulators on E2_Ubc1–Ub (PDB ID: 1FXT). The E2 catalytic cysteine (Cys) is shown as a yellow sphere. Abbreviations: PDB, Protein Data Bank; Ub, ubiquitin; Ubl, ubiquitin-like protein.

Figure 3

E3 ligases templating E2–Ubl into closed and active conformations. (a) SUMO ligase IR1 bound to E2_Ubc9 and SUMO conjugated to RanGAP, (PDB ID: 1Z5S) (b) Zoom into the E2_Ubc9 active site indicated by the box in panel a. (c) The RNF4 RING dimer with E2_Ubc5–Ub thioester mimetic bound. The second E2_Ubc5–Ub species is not shown for clarity (PDB ID: 4AP4). (d) CBL-B RING domain alone bound to E2Ubc5–Ub thioester mimetic (PDB ID: 3ZNI). Phosphorylated tyrosine (pTyr) is shown as an orange sphere. Zinc ions are represented by gray spheres, and the E2 catalytic cysteine (Cys) is shown as yellow sphere. Abbreviations: C, cysteine; D, aspartic acid; G, glycine; K, lysine; N, asparagine; PDB, Protein Data Bank; Q, glutamine; RING, really interesting new gene; RNF4, ring finger protein 4; S, serine; SUMO, small ubiquitin-like modifier protein; T, threonine; Ub, ubiquitin; Ubl, ubiquitin-like protein; Y, tyrosine.

Figure 4

Structures of the RBR E3 Ligases Parkin and HARI in Autoinhibited State. (a) Structure of Parkin (PDB IDs: 4K95 and 4K7D). (b) Structure of HHARI (PDB ID: 4KBL). Zinc ions are represented by gray spheres and the catalytic cysteines (Cys) on the RING2 domains are shown as yellow spheres. Abbreviations: IBR, in-between-RING; REP, repressor element helix; RING, really interesting new gene; UBA, ubiquitin associated domain; Ub-Like, ubiquitin-like.

Figure 5

Homologous to E6-AP C-terminus (HECT) domain conformations. (a) Nedd4 HECT domain with E2_Ubc5–Ub thioester mimetic bound (PDB ID: 3JW0). (b) Disulfide cross-linked Nedd4 HECT domain and a Ub E3 thioester mimetic complex with noncovalent ubiquitin bound (PDB ID: 4BBN). (c) Cross-linked complex between the Rsp5 WW3 and HECT domains, Ub, and the Sna3 substrate peptide (PDB ID: 4LCD). (d) Zoomed in view of the HECT active site indicated by the box in panel c. E2 and E3 catalytic cysteines are shown as yellow spheres. “Gly” refers to the anticipated position of glycine 75 in ubiquitin, which has been mutated to cysteine in this structure for crystallization purposes. Abbreviations: Arg, arginine; Cys, cysteine; Gly, glycine; PDB, Protein Data Bank; Sub, substrate; Ub, ubiquitin.