Fig. 7
From: Structural insights into the mechanism and E2 specificity of the RBR E3 ubiquitin ligase HHARI
Modeling conformational changes accompanying thioester transfer of Ub from E2 to HHARI. a Comparison of apo and E2 bound HHARI reveal conformational changes in the IR2/RING2 region. The IBR domains of apo HHARI (PDB: 4KBL, chain B) and E2 bound HHARI were superimposed and the structures are shown as cartoon representations. The hinge 1 and hinge 2 regions are boxed and magnified views are shown to the left and right of the structure, respectively. Bold black arrows indicate the direction of the rotation resulting from conformational changes in hinges 1 and 2. Disordered linkers connecting the IBR and RING2 domain and the RING2 and Ariadne domains are shown as semitransparent spheres. b Structure-function analysis of the HHARI IR2 linker. WT, mutant HHARIΔAri proteins were used in HHARI autoubiquitination assays for the indicated time points, as described in the Methods. “PP” refers to T333P/S334P HHARI double mutant. “PPPP” refers to T333P/S334P/A338P/A339P HHARI quaternary mutant. “ΔIR2 short” and “ΔIR2 long” refer to Δ333–339 and Δ322–339 HHARI deletion mutants, respectively. c Evaluation of cis vs. trans mechanisms for HHARI activation. The indicated HHARIΔAri variants were subjected to autoubiquitination assays with UchH7 for the indicated time points, as described in the Methods. d (left) The HHARI/UbcH7-Ub structure with Ub(t) (gold), UbcH7 (magenta), and the HHARI IR2 linker (slate) and RING2 domain (light blue) shown as cartoon representations. Other HHARI domains are shown as a transparent surface representation. The disordered region linking the RING2 and Ariadne domains is shown as semitransparent light blue spheres. (right) A model of an ‘active’ HHARI/UbcH7-Ub complex was created using the structure of the HOIP/UbcH5b-Ub complex (PDB:5EDV) as a guide. The hypothetical conformational changes of the HHARI IR2 and RING2-Ariadne linkers required for the RING2 and UbcH7 domains to come in proximity during thioester transfer are indicated with semitransparent slate colored spheres. The RING2/UbcH7 interface is boxed with a magnified view shown in the inset
