Figure 4.
Vpr residues that mediate HLTF degradation. A, ribbon and stick representation of Vpr (green, taken from PDB ID, 5JK7) illustrating two separate interfaces: The substrate recruitment and DCAF1 binding interfaces. Critical residues of Vpr interfacing with DCAF1 (Arg-62 and Phe-69) or substrates, including UNG2 and HLTF (Glu-24, Arg-36, Gly-43, Tyr-47, and Asp-52) identified in this and previous reports (26, 39) are shown in stick representation. B, in vitro ubiquitination assays of HLTF-HIRAN, HLTF-LINKER and HLTF-NTD with Vpr WT, G43W/Y47V or D52A in complex with CRL4-DCAF1c. C. isothermal titration trace for DDB1-DCAF1c-Vpr with G43W/Y47V mutation binding to HLTF-NTD yielded a Kd of 4.6 ± 0.4 μm. D, HEK 293T cells were co-transfected with a constant amount of full-length HLTF and DCAF1 and increasing amounts of Vpr WT or G43W/Y47V expression plasmids. All experiments were performed three times with equivalent results.