A Small Molecule That Switches a Ubiquitin Ligase From a Processive to a Distributive Enzymatic Mechanism

Abstract

E3 ligases are genetically implicated in many human diseases, yet E3 enzyme mechanisms are not fully understood, and there is a strong need for pharmacological probes of E3s. We report the discovery that the HECT E3 Nedd4-1 is a processive enzyme and that disruption of its processivity by biochemical mutations or small molecules switches Nedd4-1 from a processive to a distributive mechanism of polyubiquitin chain synthesis. Furthermore, we discovered and structurally characterized the first covalent inhibitor of Nedd4-1, which switches Nedd4-1 from a processive to a distributive mechanism. To visualize the binding mode of the Nedd4-1 inhibitor, we used X-ray crystallography and solved the first structure of a Nedd4-1 family ligase bound to an inhibitor. Importantly, our study shows that processive Nedd4-1, but not the distributive Nedd4-1:inhibitor complex, is able to synthesize polyubiquitin chains on the substrate in the presence of the deubiquitinating enzyme USP8. Therefore, inhibition of E3 ligase processivity is a viable strategy to design E3 inhibitors. Our study provides fundamental insights into the HECT E3 mechanism and uncovers a novel class of HECT E3 inhibitors.

Figure 1

Simplified model of HECT E3-mediated protein ubiquitination. E2 ~ Ub thioester binds HECT E3 domain, followed by transthiolation reaction producing HECT E3 ~ Ub thioester. Subsequently, HECT E3 ~ Ub ligates Ub onto the lysine of the substrate, followed by polyUb chain growth. C- and N-lobes of HECT domain rotate relative to each other during the catalytic cycle.

Figure 2

(A) The hotspot interface of Nedd4-1 (blue) and Ub (gray) in the Nedd4-1:Ub complex (PDB ID 2XBB) responsible for polyUb chain elongation, with the key side chains depicted as sticks. (B) Electrophilic compounds 1–3 used in this work. Compounds 1 and 2 are specific hits, and compound 3 is an optimized covalent inhibitor of Nedd4-1. (C) Cartoon depiction of the crystal structure of Nedd4-1 bound to fragment hit 1 with key side chains and the fragment shown as spheres. (D) Close-up view of the small molecule-binding site with the key side chains depicted as sticks and colored by atom type. The 2F_o – F_c electron density map (blue mesh, contoured at 1.0 σ) is presented for Cys⁶²⁷ and 1.

Figure 3

Covalent inhibitor 3 switches Nedd4-1 from a processive to a distributive enzymatic mechanism. (A) Full length Nedd4-1 with the activating E554A mutation (150 nM) was incubated with fluorescent Flu-Wbp2 substrate (100 nM) in the presence of ATP, Ub, E1, and E2 enzymes. After 1 min, a 200-fold excess of nonfluorescent Wbp2 substrate or empty buffer was added to the reaction mixture, and further ubiquitination of Flu-Wbp2 was monitored. (B) The amount of ubiquitinated Flu-Wbp2 was plotted as a function of time. (C) The same experiment was conducted with full-length Nedd4-1 E554A covalently modified with compound 3. (D) The amount of ubiquitinated Flu-Wbp2 in (C) was plotted as a function of time.

Figure 4

Ubiquitination of Flu-Wbp2 (100 nM) by a full length Nedd4-1 (150 nM) at different time points in the presence of the deubiquitinase USP8 (200 nM) shows that distributive Nedd4-1 is inhibited by USP8, but processive Nedd4-1 is not. Lane 1: DMSO treated Nedd4-1 full length E554A; lane 2: compound 3 treated Nedd4-1 full length E554A; lane 3: Nedd4-1 full length E554A F707A; and lane 4: no ATP control.