proteins STRUCTURE O FUNCTION O BIOINFORMATICS Predicting functional residues in Plasmodium falciparum plasmepsins by combining sequence and structural analysis with molecular dynamics simulations Pedro A. Valiente, 1 Paulo R. Batista, 2 Amaury Pupo, 3 Tirso Pons, 1 Alfonso Valencia, 4 and Pedro G. Pascutti 2 * 1 Facultad de Biologı ´a, Centro de Estudios de Proteı´nas (CEP), Universidad de La Habana, Cuba 2 Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil 3 Departamento de Bioinforma ´tica, Centro de Inmunologı´a Molecular, Cuba 4 Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid E-28029, Espan ˜a INTRODUCTION Malaria remains one of the world’s biggest health problems because 500 million are infected with this disease each year and it is responsible for about one million deaths annually. 1 The disease is caused by parasites from the genus Plasmo- dium and in humans, it is the result of an infection by the following species: Plasmodium falciparum, Plasmodium malariae, Plasmodium ovalae, and Plasmodium vivax. Of these species P. falciparum is the most lethal and it is there- fore the main target for drug intervention. 2 Once the microbe is transmitted to humans by mosquitoes of the anopheles genus, it causes many problems, the most com- mon of which are severe, recurring fever attacks. The increas- ing resistance of malarial parasites to the existing antimalarial drugs, and in particular of P. falciparum, has focused efforts toward the discovery of more selective and potent drugs. 3 One of the critical stages of the P. falciparum life cycle during human infection is the degradation of hemoglobin, which constitutes the main source of amino acids for its own growth and maturation. 3 During the intraerythrocytic stage of the parasite’s life cycle, this protozoa consumes 75% of the hemoglobin in the infected red blood cell. 4,5 Hemoglo- bin degradation occurs within the acidic food vacuole of the parasite and it is catalyzed by aspartic, 6 cysteine, 7 and metal- lopeptidases. 8 A family of aspartic proteases known as plas- mepsins (Plm) is involved in the initial steps of the hemoglo- Additional Supporting Information may be found in the online version of this article. Grant sponsors: Cuban Ministry of High Education (MES), Structural Biology and Bio- computing Programme of the Spanish National Cancer Research Centre (CNIO), Ma- drid, Spain. Brazilian High Education Support Agency (CAPES), International Union of Biochemistry and Molecular Biology (IUBMB). Received 24 September 2007; Revised 1 February 2008; Accepted 25 February 2008 Published online 28 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.22068 *Correspondence to: Pedro G. Pascutti, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil. E-mail: pascutti@biof.ufrj.br. ABSTRACT Plasmepsins are aspartic proteases involved in the initial steps of the hemoglobin degradation pathway, a critical stage in the Plasmodium falciparum life cycle during human infection. Thus, they are attractive targets for novel therapeutic compounds to treat malaria, which remains one of the world’s biggest health problems. The three-dimensional structures available for P. falciparum plasmepsins II and IV make structure-based drug design of antimalarial compounds that focus on inhibiting plas- mepsins possible. However, the structural flexibility of the plasmepsin active site cavity combined with insufficient knowledge of the functional residues and of those deter- mining the specificity of parasitic enzymes is a drawback when designing specific inhibitors. In this study, we have combined a sequence and structural analysis with molecu- lar dynamics simulations to predict the functional resi- dues in P. falciparum plasmepsins. The careful analysis of X-ray structures and 3D models carried out here suggests that residues Y17, V105, T108, L191, L242, Q275, and T298 are important for plasmepsin function. These seven amino acids are conserved across the malarial strains but not in human aspartic proteases. Residues V105 and T108 are localized in a flap of an interior pocket and they only establish contacts with a specific non-peptide achiral in- hibitor. We also observed a rapid conformational change in the L3 region of plasmepsins that closes the active site of the enzyme, which explains earlier experimental find- ings. These results shed light on the role of V105 and T108 residues in plasmepsin specificities, and they should be useful in structure-based design of novel, selective inhibitors that may serve as antimalarial drugs. Proteins 2008; 73:440–457. V V C 2008 Wiley-Liss, Inc. Key words: malaria; aspartic protease; selectivity; compar- ative modeling; molecular dynamics; functional residues. 440 PROTEINS V V C 2008 WILEY-LISS, INC.