Schistosoma mansoni Fatty Acid Binding Protein: Specificity and Functional Control as Revealed by Crystallographic Structure ²,‡ Francesco Angelucci, § Kenneth A. Johnson, § Paola Baiocco, § Adriana E. Miele, § Maurizio Brunori, § Cristiana Valle, | Fabio Vigorosi, | Anna Rita Troiani, | Piero Liberti, | Donato Cioli, | Mo-Quen Klinkert, ⊥ and Andrea Bellelli* ,§ Department of Biochemical Sciences “A. Rossi Fanelli”, CNR Institute of Molecular Biology and Pathology and Istituto Pasteur-Fondazione Cenci Bolognetti, UniVersity of Rome “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy, CNR Institute of Cell Biology, 32 Via Ramarini, 00016 Monterotondo, Rome, Italy, and Bernhard-Nocht-Institut fu ¨r Tropenmedizin, 74 Bernhard-Nocht-Strasse, 20359 Hamburg, Germany ReceiVed July 14, 2004 ABSTRACT: Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA) and arachidonic acid (ACD), and their structures were solved at 1.85 and 2.4 Å resolution, respectively. Sm14 is a vaccine target for schistosomiasis, the second most prevalent parasitic disease in humans. The parasite is unable to synthesize fatty acids depending on the host for these nutrients. Moreover, arachidonic acid (ACD) is required to synthesize prostaglandins employed by schistosomes to evade the host’s immune defenses. In the complex, the hydrocarbon tail of bound OLA assumes two conformations, whereas ACD adopts a unique hairpin-looped structure. ACD establishes more specific interactions with the protein, among which the most important is a π-cation bond between Arg78 and the double bond at C8. Comparison with homologous fatty acid binding proteins suggests that the binding site of Sm14 is optimized to fit ACD. To test the functional implications of our structural data, the affinity of Sm14 for 1,8-anilinonaphthalenesulfonic acid (ANS) has been measured; moreover the binding constants of six different fatty acids were determined from their ability to displace ANS. OLA and ACD exhibited the highest affinities. To determine the rates of fatty acid binding and dissociation we carried out stopped flow kinetic experiments monitoring displacement by (and of) ANS. The binding rate constant of ligands is controlled by a slow pH dependent conformational change, which we propose to have physiological relevance. Schistosomiasis is the second most prevalent parasitic disease worldwide and affects more than 200 million people in developing countries. Schistosomes are parasitic trema- todes whose complex life cycle involves an intermediate host (a freshwater snail) and man as the definitive host. Adult schistosomes live in the mesenteric or perivesical veins of their definitive host and uptake their nutrients directly from the host’s blood. The proteins that participate in the uptake and metabolism of fatty acids and their derivatives may constitute a possible target for therapy or vaccination, given the numerous and important roles played by these compounds. To briefly review this subject, we remark that the parasite lacks the metabolic pathways required for the biosynthesis of sterols and lipids; hence it is completely dependent on the host for these substances (1). Besides being nutrients and structural components of the cell membrane, fatty acid derivatives released by schistosomes play a role in the parasite evasion from the host immune response (2). Moreover, upon contact with human skin, cercariae (the larval stage released by the intermediate host) respond to chemical stimuli, particularly medium-chain free fatty acids, to start skin invasion (3). Uptake and transport of fatty acids and other lipids in S. mansoni depend (probably to a large extent) on the fatty acid binding protein (Sm14) 1 (4). Sm14 is present in all the stages of the life cycle and is localized in the external cell layer, i.e., near the interface of the parasite/host contact (5). From this short summary an important conclusion may be drawn: interfering with fatty acid uptake or metabolism may constitute an important therapeutical approach. Accordingly, the World Health Organization selected Sm14 as one out of six antischistosome vaccine candidates for testing (6, 7); and possibly the protein may also be a drug target, since blocking of fatty acid uptake could have dramatic effects on the life ² Funding was received by the University of Rome “La Sapienza” (Progetto Ateneo 60%sanno 2003) and by the Ministero dell’Universita ` e della Ricerca Scientifica e Tecnologica of Italy (PRIN 2003). Elettra (Trieste, Italy) provided generous fellowships to K.A.J. and P.B. ‡ Sm14-ACD and Sm14-OLA have been deposited with PDB codes 1VYF and 1VYG, respectively. * To whom correspondence should be addressed. Tel: +39 06499 10236. Fax: +39 06444 0062. E-mail: andrea.bellelli@uniroma1.it. § University of Rome “La Sapienza”. | CNR Institute of Cell Biology. ⊥ Bernhard-Nocht-Institut fu ¨r Tropenmedizin. 1 Abbreviations: FABP, fatty acid binding protein; Sm14, Schisto- soma mansoni FABP; SjFABP, Schistosoma japonicum FABP; Eg- FABP1, Echinococcus granulosus FABP; I-FABP, intestinal FABP; H-FABP, heart/muscle FABP; A-FABP, adipocyte FABP; B-FABP, brain FABP; M-FABP, myelin P2 FABP; L-FABP, liver FABP; CMC, critical micellar concentration; FA, fatty acid; OLA, oleic acid; ACD, arachidonic acid; LA, linoleic acid; PA, palmitic acid; MA, myristic acid; DA, decanoic acid. 13000 Biochemistry 2004, 43, 13000-13011 10.1021/bi048505f CCC: $27.50 © 2004 American Chemical Society Published on Web 09/25/2004