The structure and functional studies of RapBA1, a model regulatory aspartyl phosphate protein

The structure of RapBA1, a regulatory aspartyl phosphate phosphatase encoded by the pXO1 megaplasmid of Bacillus anthracis, has been solved to 2.85 Å. The multimeric state of the protein was studied based on the observation of a sizeable contact area between monomers of the crystallographic dimer found in the asymmetric unit. It has been determined conclusively that the native state of the protein is dimeric via size-exclusion chromatography, analytical ultracentrifugation sedimentation velocity experiments, chemical cross-linking, and analysis of a lower resolution crystal structure for of RapBA1. Gel shift assays were performed using RapBA1 with its phosphate regulatory peptide, PhrBA1. Both pentameric and heptameric peptide were tested because the literature is currently in conflict as to the physiologically relevant length. The shifts cleanly showed that the heptamer, but not the pentamer, of RapBA1 are capable of causing a shift of band position in a native gel assay. An invariant asparagine residue was located within the proposed binding domain for the regulatory peptide. This residue was located via sequence and substructure based alignments of RapBA1 to other Rap proteins and alignment to PlcR, a superfamily member of known structure. Gel shift assays were performed using an alanine-mutant of this key residue. This mutation eliminated the gel shift of RapBA1 in the presence of the heptameric PhrBA1. The result suggests that the function of this residue, binding to the backbone of the regulatory peptide, as seen in the PlcR:PapR structure, is conserved in the Rap family. These results also validate the site of peptide binding proposed based on the structure alignment of RapBA1 to PlcR based on their TPR substructures