Figure 5.

Identification of the VASA-binding surface of GUS. (A) Locations of the site-directed mutagenesis on the B30.2/SPRY domain of GUS. The green dotted circles and labels on surface A indicate the mutations that decreased the binding affinity for the VASA peptide. The dotted circles in magenta indicate the mutations on surface B, which did not affect the binding affinity. The residues of GUS corresponding to the sequence variation and the disease-related mutations in TRIM5α, Pyrin, and MID1 are highlighted by the indicated colors. (B) ITC analysis of the interaction between GUS mutants and the VASA peptide. The titration curves and curve fittings are shown for two indicated GUS mutants and the K_D values determined for the three GUS mutants are tabulated. (C) Denaturing gel elelctrophoretic analysis of the interaction between GUS mutants and the VASA peptide. Wild-type GUS and the mutants in complex with ElonginBC (each at 10 μM) were mixed with VASA peptide (30 μM) and incubated for 2 h. The mixtures were dialyzed for one day at 4°C, and each sample in the dialysis bags was subjected to denaturing gel electrophoresis. The mutants containing the amino-acid substitution on surface A or surface B are labeled with green and magenta letters, respectively. (D) Native gel electrophoretic analysis of the interaction between GUS mutants and full-length VASA. Wild-type and mutant GUS proteins in complex with ElonginBC (each at 10 μM) were mixed with VASA (5 μM) and incubated for 4 h. The analysis of the reaction mixtures by gel electrophoresis is shown. The amino-acid substitutions in the mutant proteins are color-coded as in C. While VASA mixed with wild-type GUS migrates to a distinct location, signifying a complex formation between the two, neither reduction of the band intensity nor appearance of a new band is observable for VASA mixed with the indicated GUS mutants.