Crystal structure of human PNP complexed with hypoxanthine and sulfate ion Fernanda Canduri a,b , Valmir Fadel a,b , Marcio Vinı ´cius Bertacine Dias a , Luiz Augusto Basso c , Ma ´rio Se ´rgio Palma b,d , Dio ´ genes Santiago Santos c,e, * , Walter Filgueira de Azevedo Jr. a,b, * a Programa de Po ´ s-graduac ¸a ˜o em Biofı ´sica Molecular, Departamento de Fı ´sica, UNESP, Sa ˜o Jose ´ do Rio Preto, SP 15054-000, Brazil b Center for Applied Toxinology, Instituto Butantan, Sa ˜o Paulo, SP 05503-900, Brazil c Rede Brasileira de Pesquisas em Tuberculose, Departamento de Biologia Molecular e Biotecnologia, UFRGS, Porto Alegre, RS 91501-970, Brazil d Laboratory of Structural Biology and Zoochemistry-CEIS/Department of Biology, Institute of Biosciences, UNESP, Rio Claro, SP 13506-900, Brazil e Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biome ´ dicas, PUCRS, Porto Alegre, RS, 90619-900, Brazil Received 6 November 2004 Available online 19 November 2004 Abstract Purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme, which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. Human PNP has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Here we report the crystal structure of human PNP in complex with hypoxanthine, refined to 2.6 A ˚ resolution. The intermolecular inter- action between ligand and PNP is discussed. Ó 2004 Elsevier Inc. All rights reserved. Keywords: PNP; Synchrotron radiation; Structure; Drug design; Hypoxanthine Purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1.) catalyzes the cleavage of the glycosidic bond of ribo- and deoxyribonucleosides, in the presence of inorganic orthophosphate (P i ) as a second substrate, to generate the purine base and ribose(deoxyribose)-1-phosphate. PNP is a ubiquitous enzyme of purine metabolism that functions in the salvage pathway, thus enabling the cells to utilize purine bases recovered from metabolized pur- ine ribo- and deoxyribonucleosides to synthesize purine nucleotides [1]. Human PNP is an attractive target for drug design and it has been submitted to structure-based design [2,3]. More recently, the three-dimensional struc- ture of human PNP has been refined to 2.3 A ˚ resolution [4], which allowed a redefinition of the residues involved in the substrate binding and provided a more reliable model for structure-based design of inhibitors. The crystallographic structure is a trimer and analysis of human PNP in solution, using the integration of geo- metric docking and small-angle X-ray scattering (SAXS), confirmed that the crystallographic trimer is conserved in solution [5]. Furthermore, the crystallographic structure of human PNP complexed with guanine revealed a new phosphate site, which may be the second regulatory phosphate-binding site [6]. We have obtained the crystal- lographic structure of the complex between HsPNP and hypoxanthine (HsPNP:Hx). Ligand-binding confor- mational changes and the intermolecular interaction between Hx and PNP are discussed. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.038 * Corresponding authors. E-mail addresses: diogenes@oucrs.br (D.S. Santos), walterfa@ df.ibilce.unesp.br (W.F. de Azevedo Jr.). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 326 (2005) 335–338 BBRC