Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex

Abstract

In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 approximately Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B approximately Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

Fig 1. Structure of UbcH5B Ub-HECT^NEDD4L

a–c. 3 views, separated by 90° in y (b) and then 60° in x (c) of UbcH5B (cyan) Ub (yellow)-HECT^NEDD4L (magenta). Catalytic residues (here serines, but normally cysteines as indicated) - green spheres. d–e. Structures of HECT^SMURF2 (violet, d), UbcH7-HECT^E6AP (navy, pink, e), and HECT^WWP1 (maroon), oriented as UbcH5B Ub-HECT^NEDD4L in b. Catalytic cysteines - green spheres.

Fig 2. UbcH5B-HECT^NEDD4L N-lobe interactions

a. Structural superposition of the E2s in UbcH5B (cyan) Ub (not shown for clarity)-HECT^NEDD4L (magenta) and UbcH7-HECT^E6AP (navy, pink). Catalytic residues are green spheres. b. Autoradiogram showing time course of pulse-chase transfer of ³²P-Ub from UbcH5B (E2~Ub) to wild-type (WT) and the Y736A mutant HECT^NEDD4L (E3~Ub). c. Close-up view of interactions between UbcH5B (cyan, black labels) and HECT^NEDD4L N-lobe (magenta), oriented by 90° rotation in y relative to a. d. Close-up view of interactions between UbcH7 (navy) and HECT^E6AP (salmon), in same orientation as UbcH5B-HECT^NEDD4L in c. Oxygens are red, nitrogens blue, and salt-bridges/H-bonds dashes.

Fig 3. Ub-HECT^NEDD4L C-lobe interface

a. Close-up view of Ub (yellow)-HECT^NEDD4L (magenta). In the structure, the active site cysteines are substituted with serines, but are labeled C85* and C922* from UbcH5B and NEDD4L, respectively, to indicate native sequences. Oxygens are red, nitrogens blue, and salt-bridges/H-bonds dashes. b, c. Autoradiograms showing time course of pulse-chase transfer of ³²P-Ub from UbcH5B (E2~Ub) to GST-tagged HECT domain of NEDD4L (E3~Ub), for WT and the indicated mutant of NEDD4L (b) or Ub (c).

Fig 4. Model for E2-to-HECT E3 transthiolation

a. Structural model of UbcH5B (cyan, catalytic Cys85 green) Ub (yellow)-NEDD4L HECT domain (magenta) in a putative conformation for the transthiolation reaction. ~4° rotation about the N- and C-lobes allows NEDD4L catalytic Cys922 (green) to approach the Ub C-terminus. Intermolecular contact residues poised to influence transthiolation are represented as sticks. b. Conservation among HECTs displayed on HECT^NEDD4L surface (white – no conservation, magenta – 100% identity except catalytic Cys that is green), in model of transthiolation complex with UbcH5B~Ub. c. Autoradiograms showing time course of pulse-chase transfer of ³²P-Ub from UbcH5B (E2~Ub) to GST-tagged HECT domain of NEDD4L (E3~Ub) for WT and indicated mutants in UbcH5B (top) or NEDD4L (bottom).