BeF3- acts as a phosphate analog in proteins phosphorylated on aspartate:: Structure of a BeF3- complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF3-, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Angstrom resolution the x-ray crystal structure of the complex of BeF3- with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF3- is bound to Asp-ll with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF3-. PSP and BeF3-. CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF3- for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.