Identification of the High Affinity Mn~(2+) Binding Site of Bacteriophage #lambda# Phosphoprotein Phosphatase: Effects of Metal Ligand Mutations on Electron Paramagnetic Resonance Spectra and Phosphatase Activities

摘要

Bacteriophage #lambda# phosphoprotein phosphatase (#lambda#PP) has structural similarity to the mammalian SerfThr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metalligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of #lambda#PP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to #lambda#PP are E. coli prpA and prpB. Using the crystal structure of #lambda#PP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of #lambda#PP reconstituted with Mn~(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn~(2+) binding analysis, and phosphatase kinetics. Analysis of Mn~(2+) -bound active site mutant #lambda# PP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn~(2+) and [(Mn~(2+))_2] binding. The high affinity Mn~(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.