The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly

Abstract

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration ( = 44 ± 6 nm; = 0.020 ± 0.007 s) and substrate inhibition above 0.5 μm ( = 2.5 ± 1.3 μm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 ( = 2.0 ± 0.5 μm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 ( = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 ( = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.

Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly

“…3, lane 5), as we had observed earlier for E6AP and NEDD4-2 (31)(32)(33)(34). In addition, a small amount of anchored 125 I-polyubiquitin chains conjugated to GST-NEDD4-2 is observed at the top of the resolving gel (34) (Fig. 3, lane 5).…”

“…In the kinetic experiment of Fig. 2A, the GST moiety was left unprocessed because we had observed previously that its removal has no effect on the activity of fulllength NEDD4-2 (34), suggesting that the full-length protein forms a stable oligomer in the absence of contributions from the GST moiety. Nonlinear regression analysis of the data results in a K i ϭ 5.0 Ϯ 1.4 M, in good agreement with the K i ϭ 12 Ϯ 3 M for inhibition of E6AP by the analogous GST-E6AP⌬495 mutant (31) ( Fig.…”

“…According to this model, the initial binding of E2ϳubiquitin at a cryptic site 1, not observed in the original Huang et al (24) structure but required by the more recent kinetic data, is associated with thioester exchange to yield the HECTϳubiquitin intermediate followed by binding of a second E2ϳubiquitin at the canonical E2-binding site (site 2) to support polyubiquitin chain elongation (24,32,33). More recently, parallel studies have demonstrated this two-site kinetic mechanism in the paralogous full-length NEDD4-2HECT ligase that assembles Lys-63-linked polyubiquitin chains, suggesting conservation across the superfamily (34). A concurrent observation that the evolutionarily unrelated IpaH and SspH2 families of bacterial ubiquitin ligases exhibit an analogous mechanism suggests convergent evolution, which provides strong circumstantial support for the conservation of the proximal indexation mechanism (35).…”

Developing Small‐Molecule Inhibitors of HECT‐Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities

“…This mechanism was recently experimentally validated for WWP1 . On the other hand, kinetic analyses of E6AP and NEDD4‐2 indicate an en bloc/proximal indexation mechanism, in which a ubiquitin chain is preassembled on the ligase active site with the help of two E2 enzymes before the chain is transferred to a substrate . Together, these results are in line with the idea that different ligases follow distinct mechanisms …”

“…A conserved E2‐binding site was shown to reside on the N lobe of the HECT domain, as illustrated by crystal structures of the E2‐bound HECT domains of E6AP and NEDD4‐1 . Recently, the HECT domains of E6AP and NEDD4‐2 were proposed to contain a second E2 binding site, based on kinetic analyses and modeling approaches …”