X-ray crystal structure of human VH1-related dual specificity phosphatase.

Abstract

Protein phosphorylation is the major mechanism for signaling and regulation of cellular activities. Two major groups of enzymes that hydrolyze phosphorylated amino acid residues are the serine/threonine phosphatases, and the protein tyrosine phosphatases (PTPs). Members of an emerging family of dual specificity phosphatases (DSPs) can dephosphorylate both alkyl and aryl phosphates. Some DSPs, such as cdc25 and MAP kinase phosphatase, control the cell cycle and regulate mitogenic signal transduction. Despite limited sequence homology around the Cys-X$\sb5$-Arg active-site sequence, the two-step catalytic mechanism of the DSPs is similar to that of the PTPs. Structure determination of a DSP was undertaken to provide an understanding of the structural basis of its dual substrate specificity and insights into the biological functions of DSPs. The structure of vaccinia H1-related phosphatase (or VHR), a human DSP identified from fetal brain, was determined by X-ray crystallography. Purified, recombinant VHR was crystallized in space group $P2\sb1$ with two molecules per asymmetric unit. Its three-dimensional structure was determined to 2.8 A by the multiple isomorphous replacement method using three heavy-atom derivatives, and then refined to 2.1 A resolution with an R-factor of 17.6% (25.4% R$\rm\sb{free}).$ The VHR structure contains a single domain consisting of a five-stranded, mixed $\beta$-sheet flanked by helices, which defines a conserved structural scaffold for both DSPs and PTPs. A shallow VHR active site pocket allows for the hydrolysis of phosphorylated serine, threonine, or tyrosine residues, whereas the deeper PTP active site restricts substrate specificity to only phosphotyrosine. Positively charged crevices near the active site may explain the enzyme's preference for substrates with two phosphorylated residues. A recognition region, connecting helix $\alpha1$ to strand $\beta1,$ may determine differences in substrate specificity between VHR, the PTPs, and other DSPs. A new crystal form in space group $P2\sb12\sb12\sb1$ was obtained from cocrystallization of an inactive VHR Cys$\sp{124}\to$ Ser mutant with a phosphorylated MAP kinase-derived peptide. The VHR-peptide structure was determined by the molecular replacement method using the native VHR structure as a search model. Unfortunately, the peptide did not bind to the VHR active site in this crystal form. Alternative approaches are suggested for future VHR-peptide cocrystallization.