How to Inactivate Human Ubiquitin E3 Ligases by Mutation

Abstract

E3 ubiquitin ligases are the ultimate enzymes involved in the transfer of ubiquitin to substrate proteins, a process that determines the fate of the modified protein. Numerous diseases are caused by defects in the ubiquitin-proteasome machinery, including when the activity of a given E3 ligase is hampered. Thus, inactivation of E3 ligases and the resulting effects at molecular or cellular level have been the focus of many studies during the last few years. For this purpose, site-specific mutation of key residues involved in either protein interaction, substrate recognition or ubiquitin transfer have been reported to successfully inactivate E3 ligases. Nevertheless, it is not always trivial to predict which mutation(s) will block the catalytic activity of a ligase. Here we review over 250 site-specific inactivating mutations that have been carried out in 120 human E3 ubiquitin ligases. We foresee that the information gathered here will be helpful for the design of future experimental strategies.

Keywords: E3; inactivation; ligase; mutation; ubiquitin.

FIGURE 1

Mechanism of action of RING-, HECT- and RBR-type E3 ubiquitin ligases (A) Schematic representation of a RING-type ubiquitin E3 ligase. RING E3s bind both the E2-ubiquitin and the substrate to be ubiquitinated, so bringing them together allows direct conjugation of ubiquitin (Ub) on the substrate by the E2. A monomeric RING E3 ligase is shown for illustrative purposes. (B) Schematic representation of a HECT-type ubiquitin E3 ligase. Ubiquitin is transferred first to a cysteine (C) of the HECT domain through a thioester bond and then to the substrate. (C) Schematic representation of an RBR-type ubiquitin E3 ligase. Two RING domains are separated by an in-between-RING (IBR) domain. Ubiquitin is first transferred to a cysteine (C) of the second RING domain through a thioester bond and then to the substrate.

FIGURE 2

The zinc coordinating residues in RING domains. Schematic representation of the cross-brace” topology of RING domains. The RING domain contains seven conserved cysteines and one histidine (yellow) which are involved in the coordination of two atoms of zinc. The third cysteine mediates the ubiquitin transfer in the second RING domain in RBR E3 ubiquitin ligases (contour labelled in orange). Four conserved residues (green) guide the interaction with the E2 conjugating enzyme. Mutation of the last residue of the domain (dark blue), which is normally a positively charged arginine or lysine, compromises the stability of the adjacent cysteine, affecting the coordination of the zinc atom.

FIGURE 3

Mutations that alter zinc coordinating residues in RING domains. Wheel diagram showing the reported mutations in RING domains, classified first whether a unique (single) or various (multiple) residues were mutated simultaneously. Most of the inactive E3 enzymes have been obtained by mutating key residues into alanine (yellow). Lack of activity can also be acquired by mutations into serine (orange). Mutations into other residues have been also employed (white). Zinc coordinating residues of the first (pink) and the second (light pink) RING domains in RBR-type E3 ubiquitin ligases (pink) have also be modified in order to achieve inactivation. References to all the mutations shown in this figure are provided in Supplementary Table S1.

FIGURE 4

Mutations on RING- and RBR-type E3s that affect E2-interaction, domain stabilisation, protein dimerization or substrate recognition. (A) In RING-type E3 ubiquitin ligases, inactivation can be obtained by abolishing the interaction with E2 ubiquitin-conjugating enzymes (green). This has mostly been achieved by mutating the conserved 1st (I/L) and 2nd (W/I/L) hydrophobic residues indicated in Figure 2. Other mutations affecting the stabilisation of key residues of the domain (blue), dimerization or the interaction with a specific substrate also abolish the ligase activity (purple). For the stabilisation affecting mutations, those have been classified whether a unique (single) or various (multiple) residues were mutated simultaneously. References to all the mutations shown in this figure are provided in Supplementary Table S1. (B) Alignment of the RING domains of the RING-type E3 ligases involved in E2-interacting and stabilisation mutations within the RING domain. Conserved amino acids are highlighted in yellow and orange, respectively, for the Zn-coordinating Cys and His residues, and in green for the E2-interacting residues. The conserved positively charged residues at the end of the RING domain are highlighted in blue. Mutated E2-interacting residues are shown in bold and underlined. Mutated residues involved in stabilisation are shown in bold. Mutated residues involved in dimerisation are underlined and shadowed.

FIGURE 5

Inactivation of HECT- and RBR-type E3 ligases by mutation of the catalytic cysteine. (A) Mutation of the catalytic cysteine into an alanine (light pink), a serine (pink) or another residue (white) abolishes transference of ubiquitin onto the substrate. Inactivation of the catalytic cysteine of RBR-type E3 ubiquitin ligases is obtained by mutation of the third conserved cysteine in the second RING domain. (m) indicates that this mutation has been done in the mice homolog of the protein. References to all the mutations shown in this figure are provided in Supplementary Table S1. (B) Alignment of the HECT domains of the HECT-type E3 ligases. The conserved catalytic cysteine is highlighted in pink. (C) Alignment of the RING2 domains of the RBR-type E3 ligases. Conserved zinc-coordinating cysteines and histidine on the second RING domain of mutated RBR E3 ligases are highlighted in yellow and the mutated catalytic cysteine is highlighted in pink.