Low-resolution structure and fluorescence anisotropy analysis of protein tyrosine phosphatase η catalytic domain

The rat protein tyrosine phosphatase η, rPTPη, is a class I "classical" transmembrane RPTP, with an intracellular portion composed of a unique catalytic region. The rPTPη and the human homolog DEP-1 are downregulated in rat and human neoplastic cells, respectively. However, the malignant phenotype is reverted after exogenous reconstitution of rPTPη, suggesting that its function restoration could be an important tool for gene therapy of human cancers. Using small-angle x-ray scattering (SAXS) and biophysical techniques, we characterized the intracellular catalytic domain of rat protein tyrosine phosphatase η (rPTPηCD) in solution. The protein forms dimers in solution as confirmed by SAXS data analysis. The SAXS data also indicated that rPTPηCD dimers are elongated and have an average radius of gyration of 2.65 nm and a Dmax of 8.5 nm. To further study the rPTPηCD conformation in solution, we built rPTPηCD homology models using as scaffolds the crystallographic structures of RPTPα-D1 and RPTPμ-D1 dimers. These models were, then, superimposed onto ab initio low-resolution SAXS structures. The structural comparisons and sequence alignment analysis of the putative dimerization interfaces provide support to the notion that the rPTPηCD dimer architecture is more closely related to the crystal structure of autoinhibitory RPTPα-D1 dimer than to the dimeric arrangement exemplified by RPTPμ-D1. Finally, the characterization of rPTPηCD by fluorescence anisotropy measurements demonstrates that the dimer dissociation is concentration dependent with a dissociation constant of 21.6 ± 2.0 μM. © 2007 by the Biophysical Society.