Conformational basis for substrate recruitment in protein tyrosine phosphatase 10D.
The coordinated activity of protein tyrosine phosphatases (PTPs) is crucial for
the initiation, modulation, and termination of diverse cellular processes. The
catalytic activity of this protein depends on a nucleophilic cysteine at the
active site that mediates the hydrolysis of the incoming phosphotyrosine
substrate. While the role of conserved residues in the catalytic mechanism of
PTPs has been extensively examined, the diversity in the mechanisms of substrate
recognition and modulation of catalytic activity suggests that other, less
conserved sequence and structural features could contribute to this process.
Here we describe the crystal structures of Drosophila melanogaster PTP10D in the
apo form as well as in a complex with a substrate peptide and an inhibitor.
These studies reveal the role of aromatic ring stacking interactions at the
boundary of the active site of PTPs in mediating substrate recruitment. We note
that phenylalanine 76, of the so-called KNRY loop, is crucial for orienting the
phosphotyrosine residue toward the nucleophilic cysteine. Mutation of
phenylalanine 76 to leucine results in a 60-fold decrease in the catalytic
efficiency of the enzyme. Fluorescence measurements with a competitive
inhibitor, p-nitrocatechol sulfate, suggest that Phe76 also influences the
formation of the enzyme-substrate intermediate. The structural and biochemical
data for PTP10D thus highlight the role of relatively less conserved residues in
PTP domains in both substrate recruitment and modulation of reaction kinetics.