This Article Abstract Full Text (PDF) All Versions of this Article: biophysj.106.094961v1 92/12/4424    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Matozo, H. C. Articles by Polikarpov, I. Search for Related Content PubMed PubMed Citation Articles by Matozo, H. C. Articles by Polikarpov, I.

Low-Resolution Structure and Fluorescence Anisotropy Analysis of Protein Tyrosine Phosphatase Catalytic Domain

Huita C. Matozo ^*, Maria A. M. Santos ^*, Mario de Oliveira Neto ^*, Lucas Bleicher ^*, Luís Mauricio T. R. Lima , Rodolfo Iuliano , Alfredo Fusco  and Igor Polikarpov ^* ¶

^* Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, São Carlos, Brazil; Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, Catanzaro, Italy; Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolta' di Medicina e Chirurgia, Universita' di Napoli "Federico II", Naples, Italy; and ^¶ Laboratório Nacional de Luz Síncrotron, Campinas, Brazil

Correspondence: Address reprint requests to Igor Polikarpov, Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense, 400, CEP 13566-590 São Carlos, SP, Brazil. Tel.: 55-16-3373-8088; Fax: 55-16-33739881; E-Mail: ipolikarpov{at}if.sc.usp.br.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The rat protein tyrosine phosphatase , rPTP, is a class I "classical" transmembrane RPTP, with an intracellular portion composed of a unique catalytic region. The rPTP 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 rPTP, 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 (rPTPCD) in solution. The protein forms dimers in solution as confirmed by SAXS data analysis. The SAXS data also indicated that rPTPCD 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 rPTPCD conformation in solution, we built rPTPCD homology models using as scaffolds the crystallographic structures of RPTP-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 rPTPCD dimer architecture is more closely related to the crystal structure of autoinhibitory RPTP-D1 dimer than to the dimeric arrangement exemplified by RPTPµ-D1. Finally, the characterization of rPTPCD by fluorescence anisotropy measurements demonstrates that the dimer dissociation is concentration dependent with a dissociation constant of 21.6 ± 2.0 µM.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Protein tyrosine phosphorylation is a reversible posttranslational modification involved in regulation of physiological processes as well as human health and disease. The antagonistic activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) dictate the intracellular levels of protein tyrosine phosphorylation, one of the most important eukaryotic cell signaling mechanisms (1–4). The increasing number and diversity of protein tyrosine kinases and phosphatases has been directly related to the increase in eukaryotic complexity. In recent years it became evident that, like PTKs, the PTPs play important roles in many cellular processes such as embryogenesis, organ development, tissue homeostasis, the immune system, fertilization, cell migration, and proliferation (5–7). The protein tyrosine phosphatases belong to a large group of enzymes that are subdivided into four main families based on the amino acid sequence of their catalytic domains and substrate specificity (8). The class I cysteine-based PTPs constitute by far the largest group and contain the 38 strictly tyrosine-specific PTPs. The "classical PTPs" can be further divided into transmembrane, receptor-like enzymes (RPTPs), and the intracellular, nonreceptor PTPs (NRPTPs), all sharing an intracellular catalytic domain of 240 residues. Its active site bears a signature motif (PIVVHCSAGvGRTG) where the catalytically essential cysteine and arginine are separated by five residues (9,10). The crystal structures of all known RPTP catalytic domains revealed a common architecture comprising: a globular fold that consists of an eight-stranded twisted ß-sheet flanked by four -helices on one side and another one on the opposite side; the PTP signature motif located at the bottom of the catalytic site cleft; and four loops, three of which provide residues needed for catalysis and substrate specificity (11–13).

The rat protein tyrosine phosphatase , rPTP, is a class I "classical" transmembrane RPTP, and like its human counterpart (also known as DEP-1, PTPRJ, RPTP, and CD148) it is a ubiquitous gene. In fact, rPTP is expressed in all studied tissues, including brain, liver, spleen, thyroid, and endothelial cells. The receptor-like rPTP consists of an extended extracellular region containing eight fibronectin type III motifs, a short transmembrane domain, and an intracellular portion composed of a unique catalytic region (14). Soon after its discovery, it was reported that rPTP expression is regulated by the two main thyroid regulatory pathways, suggesting it involvement in both growth and differentiation of thyroid cells (15). Further observations demonstrated that rPTP and the human homolog DEP-1 are downregulated in rat and human neoplastic cells, respectively. However, the malignant phenotype can be reverted after exogenous reconstitution of rPTP through increasing levels of cell cycle inhibitor p27^Kip1 and dephosphorylation of PLC1, a substrate of DEP-1 (16–19). It was also demonstrated that the PTP protein is capable of binding to c-Src in living cells. The dephosphorylation of the negative regulatory tyrosine (Tyr-529 of the c-Src family protein tyrosine kinases) increases c-Src tyrosine kinase activity in malignant rat thyroid cells stably transfected with rPTP (20). Besides, the implication of PTP as a possible candidate for the mouse colon-cancer susceptibility locus, Scc1, came to reinforce the idea that restoration of rPTP function could be a useful tool for gene therapy of human cancers (21–23).

Although the process of RPTP activity regulation remains elusive, one of the proposed structural mechanisms involves formation of an autoinhibitory dimeric quaternary structure. Structural evidence supporting such a mechanism came from the crystal structure of RPTP-D1 (24). RPTP-D1 was crystallized as a dimer with two active sites facing each other and the amino-terminal helix-turn-helix wedge-like segment of each monomer blocking the opposing active site (24). In addition, functional inhibition of RPTPs was observed in ligand-induced dimerization of EGFR/CD45, a chimeric protein containing the extracellular domain of the epidermal growth factor receptor (EGFR), and the intracellular domain of the RPTP, CD45 (25), as well as in the full-length mutants of RPTP assembled as stable disulphide-bonded homodimers (26,27). By contrast, the crystal structures of both RPTPµ-D1 that shares 46% sequence identity with RPTP-D1 reveal completely different dimer formation. The RPTPµ-D1 dimer has neither the catalytic sites nor the amino-terminal helix-turn-helix segment participating in protein-protein interactions (28). Taking these results together one can conclude that although dimerization seems likely to be of physiological relevance in blocking the biological activity of RPTP and CD45, it might not be a general mechanism of RPTP inhibition.

In this study, we conducted a structural and biophysical characterization of rPTP catalytic domain with use of small angle x-ray scattering (SAXS), homology modeling, amino acid sequence alignment, and fluorescence anisotropy techniques, aiming to determine the protein molecular shape and its oligomeric state in solution and to shed more light onto the putative autoinhibitory mechanism that might control rPTP enzymatic activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Protein expression and purification
The catalytic domain of rPTP has been subcloned, expressed, and purified as described in Santos et al. (29). Briefly, the cloned construct contained the open reading frame of the intracellular region of rPTP (W875-A1216) fused at its N-terminus with the 34 additional amino acid residues (MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS) derived from pET-28a(+) vector that included the cluster of six histidine residues for protein purification by metal affinity chromatography. BL21 (DE3) cells harboring the plasmid containing rPTPCD insert were grown at 30°C in 2x YT plus 50 µg/ml kanamycin with shaking until the absorbance at 600 nm reached 0.6–0.8. At this point, 0.5 mM isopropyl-ß-D-thiogalactopyranoside was added to induce His⁶-rPTPCD expression and cells were incubated at 30°C for 4 h. The induced bacteria were harvested by centrifugation at 6000 x g in a Sorvall RC-5C Plus centrifuge at 4°C for 20 min. Bacterial pellets were ressuspended in lysis buffer (50 mM sodium phosphate buffer, pH 7.8; 100 mM NaCl; 10% glycerol; 10 mM imidazol; 2 mM ß-mercaptoethanol) containing 1 mM phenylmethanesulfonyl fluoride and 0.5 mg/ml of lysozyme (Sigma, St. Louis, MO). The suspension was incubated on ice for 30 min to lyse cells. The lysate was further disrupted by sonication to reduce viscosity. Centrifugation was done at 14,000 x g for 1 h to obtain the clear crude protein preparation. Talon Superflow resin (Clontech, Palo Alto, CA), preequilibrated with equilibration buffer (50 mM sodium phosphate buffer, pH 7.8; 300 mM NaCl; 10% glycerol; 10 mM imidazol; 2 mM ß-mercaptoethanol) was mixed with the clear lysate and left rotating at 4°C for 1 h. The mixture of resin and supernatant was mounted into a c16/10 glass column (Amersham Biosciences, Uppsala, Sweden) and connected to a high-performance liquid chromatography ÄKTA purifier (Amersham Biosciences). The tightly bound proteins were eluted with buffer containing 50 mM sodium phosphate buffer, pH 7.8; 50 mM NaCl; 10% glycerol; 300 mM imidazol; 2 mM ß-mercaptoethanol. The His⁶-rPTPCD was further purified by size exclusion chromatography on a Superdex 200 HL 26/60 column (Amersham Biosciences) using as eluent Hepes buffer (20 mM Hepes, pH 7.8; 200 mM NaCl; 5% glycerol; 1 mM dithiothreitol). All purification procedures were carried out at 4°C. Soluble His⁶-rPTPCD (molecular weight of 43,000) was concentrated to 1 mg/ml and incubated with 0.5 units/ml bovine thrombin protease for 18 h at 18°C followed by dialysis against Hepes buffer. The thrombin cleaved rPTPCD (molecular weight of 41,000) was, then, frozen in liquid nitrogen and stored at –80°C (29).

Protein concentration measurements
Protein concentration was determined by the method of Bradford (30) with bovine serum albumin (Sigma) as reference standard, using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).

Small-angle x-ray scattering measurements and scattering data analysis
Small-angle x-ray scattering experiments were carried out at the SAS beam line on the Brazilian National Synchrotron Light Laboratory (Campinas, Sao Paulo, Brazil), using a one-dimensional position-sensitive detector (31). SAXS data were collected at a wavelength 0.148 nm for sample-detector distance of 1046.3 mm to give a q-range from 0.28 nm^–1 to 3.40 nm^–1 (q = 4*sin/, where 2 is the scattering angle). rPTPCD concentrations between 3 and 13 mg/ml were prepared in Hepes containing 200 mM NaCl and maintained at a constant temperature of 4°C during the measurements. The scattering curves of the protein solutions and buffers were collected in several successive frames of 100 s each to avoid radiation-induced protein damage. The data reduction included normalization of the one-dimension scattered data to the intensity of the transmitted incident beam; correction for the detector response; and subtraction of the scattering of the buffers. After scaling all scattered curves for protein concentration, the composed scattering curves were constructed by merging the low angle data (<0.85 nm^–1) obtained at 3 mg/ml with the high angle data, measured at 13 mg/ml, which were, then, used in ab initio model reconstruction.

Guinier analysis (32) was applied to determine the radii of gyration (R_g) of rPTPCD in solution. The R_g and the scattered intensity, I(q), were inferred, respectively, from the slope and the intercept of the linear fit of ln[I(q)] vs. q² in the q-range q x Rg < 1.0. The same parameter was also obtained from the theoretical fit of the merged curve by the indirect Fourier transform program GNOM (33), which also evaluated the distance distribution function, p(r), of rPTPCD. The maximum dimension, D_max, was estimated from the p(r) function as the distance, r, where p(r) goes down to zero.

Ab initio molecular shape determination and DAMAVER program
Ab initio models were calculated using the well-established reconstruction method DAMMIN, to determine the conformation in solution of rPTPCD (34). Using the simulated annealing method, the program searches for the best dummy atom model (DAM) that fits the experimental data through minimizing the discrepancy function, f(X), between the calculated and experimental curves. A looseness penalty ensures the compactness and connectivity of the solution, Eq. 1, where > 0 is the positive weight of the looseness penalty, P(X).

(1)

The overall shape of the protein was restored from the complete range of q (0.28 nm^–1 < q < 3.40 nm^–1), which allowed a reasonable resolution for the discrete DAM. In each of 20 independent low-resolution reconstructions of molecular shape, a sequence of 87 ± 3 independent runs was carried out to obtain the most probable model without imposing any symmetry constrains. During this procedure, a constant was subtracted from the experimental data to ensure that the intensity at higher angles decays as q^–4 following Porod's law for the homogeneous particles (35). The value of the constant was derived automatically from the outer part of the curve by linear fitting in coordinates q⁴I(0) versus q⁴ by the program DAMMIN.

DAMAVER was used for automated analysis and averaging of multiple reconstructions, permitting both to analyze the stability of the reconstruction convergence and to yield the most probable particle model (36,37).

Homology model building of rPTPCD
The coordinate sets for the monomeric and dimeric crystallographic structures of the catalytic domain (D1) of mouse RPTP and human RPTPµ were obtained from the Protein Data Bank ((PDB) codes 1YFO and 1RPM, respectively) (24,28). The R_g, D_max, and scattering curves were calculated from the dimeric structures by the program CRYSOL (38), taking into account the influence of the hydration shell.

Computer models for the three-dimensional (3D) structure of rPTPCD were constructed using homology modeling, which requires available 3D structures as templates. The mouse protein tyrosine phosphatase has 43.21% identity to the rPTPCD sequence, and it was selected as the template for homology model generation with the Modeller program (39). We produced 100 models, each of which was validated using the programs Procheck (40,41), Whatcheck (42), and Verify3D (43,44). The best model was chosen using as criteria the energy function calculated by the Modeller program, the Ramachandran plot, and absence of structural anomalies. The best homology model was superimposed with the dimeric crystallographic structures of RPTP-D1 and RPTPµ-D1 using SUPCOMB (36), which computes and carries out best-matching alignment of the 3D models. The same program was used for superpositions of high-resolution models with ab initio low-resolution structures derived from experimental solution-scattering data.

Fluorescence anisotropy measurements
Fluorescence anisotropy experiments were carried on the ISS PC1 spectrofluorimeter equipped with Glan-Thompson polarizers (ISS, Champaign, IL). rPTPCD at a concentration of 0.22 mM was labeled by incubation with 1 mM fluorescein isothiocyanate ((FITC) Sigma) in Hepes buffer (20 mM Hepes, pH 7.8; 200 mM NaCl; 5% glycerol; 1 mM DTT), at 4°C for 3 h. Free FITC was separated from labeled rPTPCD using Hitrap desalting column (GE Healthscience, Madison, WI) equilibrated with Hepes buffer. Protein labeling was confirmed by absorbance spectrophotometry, showing both protein and fluorescein contributions in the absorption spectra. The labeling efficiency was of 0.24 mol fluorescein per mol protein, as calculated from

(2)

where A²⁸⁰ and A⁴⁹⁴ are the absorption at 280 and 494 nm, respectively, = 68,000 M^–1cm^–1, and = 20,000 M^–1cm^–1 are the extinction coefficient for protein-bound FITC at 280 and 494 nm, respectively (45,46). = 63,050 M^–1cm^–1 is the extinction coefficient for rPTP catalytic domain at 280 nm calculated on the basis of its amino acid sequence (residues W875-A1216 of rPTP; "Protein expression and purification" section) assuming six tryptophan residues, 20 tyrosine residues, and at most two cystines (47). The low efficiency of labeling, probably due to the pH and temperature conditions used in the reactions, is advantageous because it diminishes the chances of self-transfer between fluorophores on the same protein molecule.

Fluorescein-rPTPCD at a concentration of 10 nM was titrated with unlabeled rPTPCD at 20°C in Hepes buffer. Fluorescence anisotropy of the labeled solution of rPTPCD was excited at 480 nm and emission measured through a band-pass filter with a cutoff of 50% at 515 nm, as previously described (48). For each concentration of unlabeled protein, the data point represents the mean of at least five measurements after stabilization. The resulting anisotropy change was used to calculate the dissociation equilibrium constant by adjusting Eq. 3 to the experimental data:

(3)

where f_d is the fraction of protein dimers and K_d is the dissociation constant. As the association led to a discrete increase in fluorescence intensity (40%), we applied correction:

(4)

where A_obs, A_m, and A_d are, respectively, the rPTPCD-labeled anisotropy values observed at a given concentration of unlabeled rPTPCD, for the monomeric rPTPCD and dimeric rPTPCD (49). The corrections performed on changes in fluorescence intensity make use of the total fluorescence intensity I_T directly provided by the instrument software, according to:

(5)

where I_|| and I are, respectively, the time-independent steady-state values for fluorescent intensity polarized parallel and perpendicular to that of the excitation beam. Binding data were analyzed using SigmaPlot 2002 Windows Version 8.0.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Small-angle x-ray scattering measurements demonstrate that rPTPCD is capable of dimer formation
The biological activity of the purified rPTPCD was tested in vitro by hydrolysis assay. The results demonstrated that rPTPCD was active and able to hydrolyze p-nitrophenyl phosphate (29). To obtain information about the size, molecular shape, and oligomeric state of rPTPCD in solution, we submitted the protein to small-angle x-ray scattering analysis. rPTPCD x-ray scattering curve is shown in Fig. 1 A. The Guinier analysis, in the q²-range from 0.08 to 0.24 nm^–2, reproducibly gave estimates of 2.59 nm for the radius of gyration of rPTPCD. The linearity of the Guinier plot indicated that rPTPCD preparation was monodisperse and constituted mainly by unique oligomeric species (Fig. 1 A, inset).

View larger version (20K): [in this window] [in a new window]   FIGURE 1  Small angle x-ray scattering curves. (A) Experimental solution scattering curve of rPTPCD and results of the fitting procedures. Intensity curves from rPTPCD 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 RPTP-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 rPTPCD. The p(r) values of rPTPCD (open squares) computed from the experimental scattering curves by the indirect Fourier transform program GNOM (33). The maximum dimension (Dmax) is equal to 8.50 ± 1.00 nm. The black solid curve corresponds to p(r) derived from dimer homology model based on the RPTP-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 RPTP-D1 dimer is clearly more similar to the experimentally derived p(r) for rPTPCD than the one calculated on the basis of RPTPµ-D1 dimer model.

Ab initio molecular shapes of rPTPCD and its high-resolution models

View larger version (72K): [in this window] [in a new window]   FIGURE 2  Three orthogonal stereoviews of the ab initio low-resolution rPTPCD structure superposed with the dimeric homology model of rPTPCD based on the crystallographic structure of RPTP-D1. The figure was prepared using PyMOL (DeLano Scientific, San Carlos, CA; http://www.pymol.org).

View larger version (71K): [in this window] [in a new window]   FIGURE 3  Three orthogonal stereoviews of the homology model of the putative rPTPCD dimer based on the crystallographic structure of RPTPµ-D1 superposed on the low-resolution ab initio rPTPCD molecular envelope. The figure was prepared using PyMOL (DeLano Scientific).

View larger version (65K): [in this window] [in a new window]   FIGURE 4  Sequence alignment for RPTP-D1, RPTPµ-D1, and PTPCD. The sequence of PTPCD corresponds to the intracellular domain of the protein studied in current work (residues W875–A1216). The sequences of RPTP-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 (RPTP-D1) and black (RPTPµ-D1). Residues in rPTPCD amino acid sequence are gray when they match the correspondent residues of the RPTP-D1 dimerization interface; they are black when identical to residues at the dimerization interface of RPTPµ-D1.

View larger version (64K): [in this window] [in a new window]   FIGURE 5  Crystallographic structures of RPTP-D1 and RPTPµ-D1. The dimerization interfaces of RPTP-D1 and RPTPµ-D1 are shown as surfaces. (A) The residues of RPTP-D1 that are conserved in rPTPCD 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 rPTPCD 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).

Fluorescence anisotropy measurements provide an equilibrium dissociation constant of the rPTPCD dimers

View larger version (13K): [in this window] [in a new window]   FIGURE 6  Equilibrium fluorescence anisotropy. (A) Fluorescence spectra of buffer and fluorescein-labeled rPTPCD. (B) Measurements of rPTPCD 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).

A number of RPTPs, as well as RPTKs, are known to function as transmembrane dimers and dimerization could be a common phenomenon for RPTPs in general (26,27,56). Although the dimeric interaction seen for soluble rPTPCD is 22 µM, this affinity is expected to be enhanced substantially for the full-length receptors in the cell membrane. In fact, both local protein concentrations and monomer-monomer affinities could be significantly higher for the functional rPTP dimers bound to the membrane of cell. Under these settings two extracellular domains of PTP, each composed of eight fibronectin type 3 motifs, will also contribute to PTP dimer formation. Membrane anchoring, which restricts both PTP translational movement in the direction perpendicular to the membrane surface, and its rotational freedom around the axes within the membrane plane, will lead to further increase in association affinity of PTP dimer. It has been argued that the binding affinities of the molecules in two-dimensional diffusion case are significantly (orders of magnitude) higher than observed in solution (57,58). Therefore, affinities of dimers association are expected to be substantially enhanced, and equilibrium dissociation constants significantly diminished, under conditions of the full-length rPTPs anchored in the membrane.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Dimerization is a well-established regulatory mechanism for activation of RPTKs and is likely to be involved in regulation of RPTPs. Although many protein tyrosine phosphatases have two intracellular domains, only the plasma membrane-proximal domain (D1) has significant catalytic activity. Dimeric crystallographic structure of RPTP-D1 revealed the presence of an NH₂-terminal helix-turn-helix segment interacting with the opposing monomer, in such a manner that both catalytic sites in the dimer are blocked (24). On the other hand, it has been shown that dimerization of Sap-1/PTPRH, a single intracellular domain RPTP, does not occur through its catalytic domain (59). Given a fact that rPTP also has a unique catalytic domain, we studied its behavior in solution using synchrotron small-angle x-ray scattering, homology modeling, sequence alignment, and fluorescence anisotropy. Here, we provide the first ab initio low-resolution structure of rPTPCD in solution restored at 1.85-nm resolution from synchrotron small-angle x-ray scattering data. The results from SAXS reveal that rPTPCD forms dimers in solution. The fluorescence anisotropy demonstrates that the dimeric form of rPTPCD is the predominant species in solution with a monomer-dimer equilibrium dissociation constant of 21.6 ± 2.0 µM and a corresponding equal to 6.2 kcal/mol. Ab initio SAXS model building, homology modeling, and sequence analysis data of the putative rPTPCD dimeric interfaces taken together strongly indicate that rPTP dimeric architecture might be closely related to that of RPTP-D1. It is tempting to speculate that dimerization of rPTPCD could be involved in the self-inhibition of its enzymatic activity. In this case the autoinhibitory mechanism would occur through the N-terminal wedge region-active site interactions as proposed for RPTP-D1, a hypothesis amenable to further site-directed mutagenesis analysis, and in vivo and in vitro functional studies.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grants No. 99/03387-4 and No. 06/00182-8) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Submitted on August 12, 2006; accepted for publication February 22, 2007.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
1. Hunter, T. 1987. A 1001 protein-kinases. Cell. 50:823–829.[CrossRef][Medline]

2. Hunter, T. 1995. Protein-kinases and phosphatases: the Yin and Yang of protein-phosphorylation and signaling. Cell. 80:225–236.[CrossRef][Medline]

3. Tonks, N. K., and B. G. Neel. 1996. From form to function: signaling by protein tyrosine phosphatases. Cell. 87:365–368.

4. Neel, B. G., and N. K. Tonks. 1997. Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell Biol. 9:193–204.[CrossRef][Medline]

5. Chagnon, M. J., N. Uetani, and M. L. Tremblay. 2004. Functional significance of the LAR receptor protein tyrosine phosphatase family in development and diseases. Biochem. Cell Biol. 82:664–675.[CrossRef][Medline]

6. den Hertog, J. 1999. Protein-tyrosine phosphatases in development. Mech. Dev. 85:3–14.[CrossRef][Medline]

7. Mustelin, T., R. T. Abraham, C. E. Rudd, A. Alonso, and J. J. Merlo. 2002. Protein tyrosine phosphorylation in T cell signaling. Front. Biosci. 7:918–969.[CrossRef]

8. Fauman, E. B., and M. A. Saper. 1996. Structure and function of the protein tyrosine phosphatases. Trends Biochem. Sci. 21:414–417.

9. Alonso, A., J. Sasin, N. Bottini, I. Friedberg, I. Friedberg, A. Osterman, A. Godzik, T. Hunter, J. Dixon, and T. Mustelin. 2004. Protein tyrosine phosphatases in the human genome. Cell. 117:699–711.[CrossRef][Medline]

10. Kolmodin, K., and J. Åqvist. 2001. The catalytic mechanism of protein tyrosine phosphatases revisited. FEBS Lett. 498:208–213.[CrossRef][Medline]

11. Jia, Z., D. Barford, A. J. Flint, and N. K. Tonks. 1995. Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. Science. 268:1754–1758.[Abstract/Free Full Text]

12. Stuckey, J. A., H. L. Schubert, E. B. Fauman, Z. Y. Zhang, J. E. Dixon, and M. A. Saper. 1994. Crystal structure of Yersinia protein tyrosine phosphatase at 2.5 A and the complex with tungstate. Nature. 370:571–575.[CrossRef][Medline]

13. Barford, D., A. K. Das, and M. P. Egloff. 1998. The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu. Rev. Biophys. Biomol. Struct. 27:133–164.[CrossRef][Medline]

14. Zhang, L., M. L. Martelli, C. Battaglia, F. Trapasso, D. Tramontano, G. Viglietto, A. Porcellini, M. Santoro, and A. Fusco. 1997. Thyroid cell transformation inhibits the expression of a novel rat protein tyrosine phosphatase. Exp. Cell Res. 235:62–70.[CrossRef][Medline]

15. Martelli, M. L., F. Trapasso, P. Bruni, M. T. Berlingieri, C. Battaglia, M. T. Vento, B. Belletti, R. Iuliano, M. Santoro, G. Viglietto, and A. Fusco. 1998. Protein tyrosine phosphatase-eta expression is upregulated by the PKA-dependent and is downregulated by the PKC-dependent pathways in thyroid cells. Exp. Cell Res. 245:195–202.[CrossRef][Medline]

16. Trapasso, F., R. Iuliano, A. Boccia, A. Stella, R. Visconti, P. Bruni, G. Baldassarre, M. Santoro, G. Viglietto, and A. Fusco. 2000. Rat protein tyrosine phosphatase eta suppresses the neoplastic phenotype of retrovirally transformed thyroid cells through the stabilization of p27(Kip1). Mol. Cell. Biol. 20:9236–9246.[Abstract/Free Full Text]

17. Florio, T., S. Arena, S. Thellung, R. Iuliano, A. Corsaro, A. Massa, A. Pattarozzi, A. Bajetto, F. Trapasso, A. Fusco, and G. Schettini. 2001. The activation of phosphotyrosine phosphatase eta (r-PTP eta) is responsible for the somatostatin inhibition of PC Cl3 thyroid cell proliferation. Mol. Endocrinol. 15:1838–1852.[Abstract/Free Full Text]

18. Iuliano, R., F. Trapasso, I. Le Pêra, F. Schepis, I. Samà, A. Clodomiro, K. R. Dumon, M. Santoro, L. Chiariotti, G. Viglietto, and A. Fusco. 2003. An adenovirus carrying the rat protein tyrosine phosphatase eta suppresses the growth of human thyroid carcinoma cell lines in vitro and in vivo. Cancer Res. 63:882–886.[Abstract/Free Full Text]

19. Trapasso, F., S. Yendamuri, K. R. Dumon, R. Iuliano, R. Cesari, B. Feig, R. Seto, L. Infante, H. Ishii, A. Vecchione, M. J. During, C. M. Croce, and A. Fusco. 2004. Restoration of receptor-type protein tyrosine phosphatase eta function inhibits human pancreatic carcinoma cell growth in vitro and in vivo. Carcinogenesis. 25:2107–2114.[Abstract/Free Full Text]

20. Le Pêra, I., R. Iuliano, T. Florio, C. Susini, F. Trapasso, M. Santoro, L. Chiariotti, G. Schettini, G. Viglietto, and A. Fusco. 2005. The rat tyrosine phosphatase eta increases cell adhesion by activating c-Src through dephosphorylation of its inhibitory phosphotyrosine residue. Oncogene. 24:3187–3195.[CrossRef][Medline]

21. Iuliano, R., I. Le Pera, C. Cristofaro, F. Baudi, F. Arturi, P. Pallante, M. L. Martelli, F. Trapasso, L. Chiariotti, and A. Fusco. 2004. The tyrosine phosphatase PTPRJ/DEP-1 genotype affects thyroid carcinogenesis. Oncogene. 23:8432–8438.[CrossRef][Medline]

22. Ruivenkamp, C. A., T. van Wezel, C. Zanon, A. P. M. Stassen, C. Vlcek, T. Csikos, A. M. Klous, N. Tripodis, A. Perrakis, L. Boerrigter, P. C. Groot, J. Lindeman, et al. 2002. Ptprj is a candidate for the mouse colon-cancer susceptibility locus Scc1 and is frequently deleted in human cancers. Nat. Genet. 31:295–300.[CrossRef][Medline]

23. Ruivenkamp, C. A., M. Hermsen, C. Postma, A. Klous, J. Baak, G. Meijer, and P. Demant. 2003. LOH of PTPRJ occurs early in colorectal cancer and is associated chromosomal loss of 18q12–21. Oncogene. 22:3472–3474.[CrossRef][Medline]

24. Bilwes, A. M., J. den Hertog, T. Hunter, and J. P. Noel. 1996. Structural basis for inhibition of receptor protein-tyrosine phosphatase-alpha by dimerization. Nature. 382:555–559.[CrossRef][Medline]

25. Majeti, R., A. M. Bilwes, J. P. Noel, T. Hunter, and A. Weiss. 1998. Dimerization-induced inhibition of receptor protein tyrosine phosphatase function through an inhibitory wedge. Science. 279:88–91.[Abstract/Free Full Text]

26. Jiang, G., J. den Hertog, J. Su, J. Noel, J. Sap, and T. Hunter. 1999. Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase-alpha. Nature. 401:606–610.[CrossRef][Medline]

27. Jiang, G., J. den Hertog, and T. Hunter. 2000. Receptor-like protein tyrosine phosphatase alpha homodimerizes on the cell surface. Mol. Cell. Biol. 20:5917–5929.[Abstract/Free Full Text]

28. Hoffmann, K. M. V., N. K. Tonks, and D. Barford. 1997. The crystal structure of domain 1 of receptor protein-tyrosine phosphatase mu. J. Biol. Chem. 272:27505–27508.[Abstract/Free Full Text]

29. Santos, M. A. M., S. M. Santos, H. C. Matozo, R. V. Portugal, R. Iuliano, A. Fusco, and I. Polikarpov. 2005. Expression, purification, and characterization of rat protein tyrosine phosphatase eta catalytic domain. Protein Exp. Purif. 41:113–120.[CrossRef][Medline]

30. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.[CrossRef][Medline]

31. Kellermann, G., F. Vicentin, E. Tamura, M. Rocha, H. Tolentino, A. Barbosa, A. Craievich, and I. Torriani. 1997. The small-angle x-ray scattering beamline of the Brazilian Synchrotron Light Laboratory. J. Appl. Crystallogr. 30:880–883.[CrossRef]

32. Guinier, A., and G. Fournet. 1995. Small-Angle Scattering of X-rays. John Wiley & Sons, New York.

33. Svergun, D. I. 1992. Determination of the regularization parameter in indirect-transform methods using percentual criteria. J. Appl. Crystallogr. 25:495–503.[CrossRef]

34. Svergun, D. I. 1999. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76:2879–2886.[Abstract/Free Full Text]

35. Porod, G. 1982. General theory. In Small-Angle X-ray Scattering. O. Glatter and O. Kratky, editors. Academic Press, London, UK. 17–51.

36. Kozin, M. B., and D. I. Svergun. 2001. Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34:33–41.[CrossRef]

37. Svergun, D. I., and M. H. Koch. 2002. Advances in structural analysis using small-angle scattering in solution. Curr. Opin. Struct. Biol. 12:654–660.[CrossRef][Medline]

38. Svergun, D. I., C. Barberato, and M. H. Koch. 1995. CRYSOL: a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28:768–773.[CrossRef]

39. Sali, A., and T. L. Blundell. 1993. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234:779–815.[CrossRef][Medline]

40. Laskowski, R. A., M. W. MacArthur, D. S. Moss, and J. M. Thornton. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26:283–291.[CrossRef]

41. Morris, A. L., M. W. MacArthur, E. G. Hutchinson, and J. M. Thornton. 1992. Stereochemical quality of protein structure coordinates. Proteins. 12:345–364.[CrossRef][Medline]

42. Hooft, R. W. W., G. Vriend, C. Sander, and E. E. Abola. 1996. Errors in protein structures. Nature. 381:272.[Medline]

43. Bowie, J. U., R. Luthy, and D. Eisenberg. 1991. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 253:164–170.[Abstract/Free Full Text]

44. Luthy, R., J. U. Bowie, and D. Eisenberg. 1992. Assessment of protein models with three-dimensional profiles. Nature. 356:83–85.[CrossRef][Medline]

45. www.piercenet.com/files/TR0031dh5-Calc-FP-ratios.pdf. 2007. [Online].

46. www.markergene.com/product_sheets/pis0955.pdf. 2007. [Online].

47. Pace, C. N., F. Vajdos, L. Fee, G. Grimsley, and T. Gray. 1996. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411–2423.

48. Lima, L. M. T. R., and J. L. Silva. 2004. Positive contribution of hydration on DNA binding by E2c protein from papillomavirus. J. Biol. Chem. 279:47968–47974.[Abstract/Free Full Text]

49. Malencik, D. A., and S. R. Anderson. 1984. Peptide binding by calmodulin and its proteolytic fragments and by troponin C. Biochemistry. 23:2420–2428.[CrossRef][Medline]

50. McKinney, R., L. Thacker, and G. A. Hebert. 1976. Conjugation methods in immunofluorescence. J. Dent. Res. 55:A38–A44.[Medline]

51. Xu, G.-J., and G. Weber. 1982. Dynamics and time-averaged chemical potential of proteins: importance in oligomer association. Proc. Natl. Acad. Sci. USA. 79:5268–5271.[Abstract/Free Full Text]

52. Weber, G. 1992. Protein Interactions. Chapter XIV. Chapman and Hall, New York.

53. Shore, J. D., and S. K. Chakrabarti. 1976. Subunit dissociation of mitochondrial malate dehydrogenase. Biochemistry. 15:875–879.[CrossRef][Medline]

54. Silva, J. L., C. F. Silveira, A. Correia Jr., and L. Pontes. 1992. Dissociation of a native dimer to a molten globule monomer: effects of pressure and dilution on the association equilibrium of arc repressor. J. Mol. Biol. 223:545–555.[CrossRef][Medline]

55. Moreau, V. H., A. C. da Silva, R. M. Siloto, A. P. Valente, A. Leite, and F. C. Almeida. 2004. The bZIP region of the plant transcription factor opaque-2 forms stable homodimers in solution and retains its helical structure upon subunit dissociation. Biochemistry. 43:4862–4868.[CrossRef][Medline]

56. Stoker, A. W. 2005. Protein tyrosine phosphatases and signaling. J. Endo. 185:19–33.

57. Grasberger, B., A. P. Minton, C. DeLisi, and H. Metzger. 1986. Interaction between proteins localized in membranes. Proc. Natl. Acad. Sci. USA. 83:6258–6262.[Abstract/Free Full Text]

58. Fan, Q. R., and W. A. Hendrickson. 2005. Structure of human follicle-stimulating hormone in complex with its receptor. Nature. 433:269–277.[CrossRef][Medline]

59. Walchli, S., X. Espanel, and R. H. van Huijsduijnen. 2005. Sap-1/PTPRH activity is regulated by reversible dimerization. Biochem. Biophys. Res. Commun. 331:497–502.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: biophysj.106.094961v1 92/12/4424    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Matozo, H. C. Articles by Polikarpov, I. Search for Related Content PubMed PubMed Citation Articles by Matozo, H. C. Articles by Polikarpov, I.