Despite belonging to the phosphoserine- and phosphothreonine-specific phosphoprotein phosphatase (PPP) family, -like phosphatase 2 (RLPH2) strongly prefers substrates bearing phosphorylated tyrosine residues. We solved the structures of RLPH2 crystallized in the presence or absence of sodium tungstate. These structures revealed the presence of a central domain that forms a binding site for two divalent metal ions that closely resembles that of other PPP-family enzymes. Unique structural elements from two flanking domains suggest a mechanism for the selective dephosphorylation of phosphotyrosine residues. Cocrystallization with the phosphate mimetic tungstate also suggests how positively charged residues that are highly conserved in the RLPH2 class form an additional pocket that is specific for a phosphothreonine residue located near the phosphotyrosine residue that is bound to the active site. Site-directed mutagenesis confirmed that this auxiliary recognition element facilitates the recruitment of dual-phosphorylated substrates containing a pTxpY motif.

Despite belonging to the phosphoserine-and phosphothreonine-specific phosphoprotein phosphatase (PPP) family, Arabidopsis thaliana Rhizobiales-like phosphatase 2 (RLPH2) strongly prefers substrates bearing phosphorylated tyrosine residues. We used the anomalous scattering signal from sulfur atoms in the native protein to calculate phases for modest-quality diffraction patterns measured from needle-like crystals (1.0 x 0.01 x 0.01 mm) of RLPH2 (P61; dmin = 2.2 Å; Rsym = 0.092; redundancy = 30.9; λ = 1.853 Å). As expected for a PPP-family enzyme, the structure of RLPH2 contains a central domain that forms a binding site for two divalent metal ions. Distinctive structural elements from two flanking domains contribute structural elements that suggest a novel mechanism for the selective dephosphorylation of phosphotyrosine residues. Co-crystallization with the phosphate mimetic tungstate also suggests how positively charged residues that are highly conserved in the RLPH2 class form an additional pocket that is specific for a phosphothreonine residue located near the phosphotyrosine residue that is bound to the active site. Site-directed mutagenesis confirmed that this auxiliary recognition element facilitates the recruitment of dual-phosphorylated substrates containing a pTxpY motif. The combination of an auxiliary site specific for phosphothreonine and the active-site specific for phosphotyrosine reveals a novel mechanism for the recognition of dually phosphorylated substrates that appears to be conserved in the RLPH family of plant protein phosphatases.