Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation

Abstract

We seek to develop quantitative assays of individual steps that underlie substrate ubiquitination, to gain a detailed understanding of the enzymological mechanism by which E2 and E3 collaborate to specify the synthesis of an ubiquitin chain upon substrate. We have directed our initial efforts to understanding how conjugation of Nedd8 influences the activity of the archetypal cullin‐RING ligase (CRL) SCF. Conjugation of ubiquitin‐like protein Nedd8 to cullin (i.e. neddylation) is known to be essential for the function of CRLs. Our data show that neddylation acts through multiple channels to stimulate SCF. First, it stimulates recruitment of ubiquitin‐conjugating enzyme (E2) esterified with ubiquitin (E2~Ub). Second, it helps bridge the ~50 Ågap between E2 and substrate bound to SCF to enable their reaction. Third, it facilitates formation of amide bonds in E2's active site. Combined together, these effects potently stimulate transfer of the initial ubiquitin to substrate. We propose that the initiator ubiquitin partly spans the 50 Ågap, and the impact of neddylation on transfer of subsequent ubiquitins by the E2 Cdc34 arises primarily from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires =4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation. Given the major functional impact of Nedd8 conjugation upon SCF activity, we sugget that the autoubiquitination often exhibited by other E3s has the potential to have major functional consequences on E3 activity.