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

Abstract

The rat protein tyrosine phosphatase eta, rPTPeta, is a class I "classical" transmembrane RPTP, with an intracellular portion composed of a unique catalytic region. The rPTPeta 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 rPTPeta, 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 eta (rPTPetaCD) in solution. The protein forms dimers in solution as confirmed by SAXS data analysis. The SAXS data also indicated that rPTPetaCD dimers are elongated and have an average radius of gyration of 2.65 nm and a D(max) of 8.5 nm. To further study the rPTPetaCD conformation in solution, we built rPTPetaCD homology models using as scaffolds the crystallographic structures of RPTPalpha-D1 and RPTPmicro-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 rPTPetaCD dimer architecture is more closely related to the crystal structure of autoinhibitory RPTPalpha-D1 dimer than to the dimeric arrangement exemplified by RPTPmicro-D1. Finally, the characterization of rPTPetaCD by fluorescence anisotropy measurements demonstrates that the dimer dissociation is concentration dependent with a dissociation constant of 21.6 +/- 2.0 microM.

FIGURE 1

Small angle x-ray scattering curves. (A) Experimental solution scattering curve of rPTPηCD and results of the fitting procedures. Intensity curves from rPTPηCD are shown as logarithm (log I) versus the momentum transfer q scale, with an inset containing the correspondent Guinier plots (log I vs. q²). The desmeared experimental curve (open squares) denotes the SAXS data and the error bars indicate the standard uncertainty in the measurement. The dashed curve corresponds to the best-fit model produced by DAMMIN (34). The black solid line is the theoretical scattering intensity from the dimer homology model based on the RPTPα-D1 (PDB code, 1YFO). The gray solid line is the scattering intensity computed from the dimer homology model based on the RPTPμ-D1 crystal structure (PDB code, 1RPM). (B) Distance distribution function of rPTPηCD. The p(r) values of rPTPηCD (open squares) computed from the experimental scattering curves by the indirect Fourier transform program GNOM (33). The maximum dimension (D_max) is equal to 8.50 ± 1.00 nm. The black solid curve corresponds to p(r) derived from dimer homology model based on the RPTPα-D1 dimer. The p(r) derived from dimer homology model based on the dimer of RPTPμ-D1 is given as a gray solid line. The distance distribution function for RPTPα-D1 dimer is clearly more similar to the experimentally derived p(r) for rPTPηCD than the one calculated on the basis of RPTPμ-D1 dimer model.

FIGURE 2

Three orthogonal stereoviews of the ab initio low-resolution rPTPηCD structure superposed with the dimeric homology model of rPTPηCD based on the crystallographic structure of RPTPα-D1. The figure was prepared using PyMOL (DeLano Scientific, San Carlos, CA; http://www.pymol.org).

FIGURE 3

Three orthogonal stereoviews of the homology model of the putative rPTPηCD dimer based on the crystallographic structure of RPTPμ-D1 superposed on the low-resolution ab initio rPTPηCD molecular envelope. The figure was prepared using PyMOL (DeLano Scientific).

FIGURE 4

Sequence alignment for RPTPα-D1, RPTPμ-D1, and PTPηCD. The sequence of PTPηCD corresponds to the intracellular domain of the protein studied in current work (residues W875–A1216). The sequences of RPTPα-D1 and RPTPμ-D1 are of the polypeptide chains structurally defined in PDBs 1YFO and 1RPM, respectively. The residues at the dimerization interfaces are marked in gray (RPTPα-D1) and black (RPTPμ-D1). Residues in rPTPηCD amino acid sequence are gray when they match the correspondent residues of the RPTPα-D1 dimerization interface; they are black when identical to residues at the dimerization interface of RPTPμ-D1.

FIGURE 5

Crystallographic structures of RPTPα-D1 and RPTPμ-D1. The dimerization interfaces of RPTPα-D1 and RPTPμ-D1 are shown as surfaces. (A) The residues of RPTPα-D1 that are conserved in rPTPηCD are shown in black; the nonconserved residues are painted in gray. (B) The same molecule as in panel A rotated by 180° across the vertical axis. (C) The residues of RPTPμ-D1 that are conserved in rPTPηCD are depicted in black; the ones that are nonconserved are given in gray. (D) The same molecule as in panel C rotated by 180° across the vertical axis. The figure was prepared using PyMOL (DeLano Scientific).

FIGURE 6

Equilibrium fluorescence anisotropy. (A) Fluorescence spectra of buffer and fluorescein-labeled rPTPηCD. (B) Measurements of rPTPηCD dimer-monomer reaction were performed as described in experimental procedures. The results of three independent experiments are shown as raw anisotropy. (Inset) Fraction of association calculated from anisotropy values properly corrected for changes in fluorescence intensity. Solid line corresponds to the best fit of Eq. 3 to data, which yields a dissociation constant of 21.6 ± 2.0 μM (r² = 0.988).