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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.

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.