Extended Data Figure 5: The kink in the pUBH.

a–d, To understand whether the straight pUBH in PhPARKIN was a consequence of a helix-favouring mutation of Gly319 (HsPARKIN) to Ala (Ala321 in PhPARKIN), the HsPARKIN(ΔUbl) G319A mutant was crystallized and a structure determined at 2.35 Å resolution (see Extended Data Fig. 1e and Methods). a, Superposition of HsPARKIN(ΔUbl) G319A and wild-type HsPARKIN(ΔUbl) (PDB 4BM9 (ref. 14)). The structures are virtually identical, both containing a kinked helix. b, c, Electron density detail of the kinked helix for the mutant (b) and wild type (c), shown in stereo representation, with 2|F_o| − |F_c| density contoured at 1σ. The Cβ atom of Ala319 is clearly defined in electron density and does not induce helix straightening in this crystallographic setting. d, Comparison of pUBH helices in HsPARKIN(ΔUbl) G319A (left), HsPARKIN(ΔUbl) wild type (second from left), superposition of the two (second from right), and for comparison the straight pUBH in PhPARKIN–pUb. e, f, Structure of the RBR E3 ligase HHARI (e) in an autoinhibited form (PDB 4KBL (ref. 23)) showing an entirely different RBR module as compared to full-length RnPARKIN (PDB 4K95 (ref. 16)) in f. RING1, IBR, RING2 domains are coloured as for PARKIN in Fig. 1, and other domains (UBA-like domain and Ariadne domain in HHARI) are coloured in red. Interestingly, the pUBH equivalent helix in HHARI is kinked at a similar position as compared to mammalian PARKIN. g, Superposition of structures from e and f on their RING1 domains. In HHARI, the pUBH-equivalent helix kinks in a different manner as compared to HsPARKIN. h, In HHARI, the kinked helix seems to be stabilized by an interaction in cis with the UBA-like domain that binds NEDD8 (ref. 37). The sequence of the helix does not contain Gly residues, but is kinked at a Thr residue (Thr263). It will be interesting to see whether helix straightening occurs in active forms of HHARI that can be induced by binding NEDD8-modified cullins³⁷.