Heme Binding to the Histidine-Rich Protein II from Plasmodium falciparum † Eric L. Schneider ‡ and Michael A. Marletta* ,‡,§,| Department of Chemistry, Department of Molecular and Cell Biology, and DiVision of Physical Biosciences, Lawrence Berkeley National Laboratory, UniVersity of California, Berkeley, Berkeley, California 94720-1460 ReceiVed July 6, 2004; ReVised Manuscript ReceiVed October 5, 2004 ABSTRACT: The histidine-rich protein II (HRP II) from Plasmodium falciparum has been implicated in the formation of hemozoin, a detoxified, crystalline form of ferric protoporphyrin IX (Fe 3+ -PPIX) produced by the parasite. Fe 3+ -PPIX titrations coupled with quantitative amino acid analysis showed that HRP II binds 15 Fe 3+ -PPIX molecules per 30 kDa monomer. Circular dichroism spectroscopy was used to probe the secondary structure of HRP II with and without bound Fe 3+ -PPIX. These studies have revealed large changes in the secondary structure with Fe 3+ -PPIX binding, changing from a random coil in the absence of Fe 3+ -PPIX to a more ordered helical structure in the presence of Fe 3+ -PPIX. The Fe 3+ -PPIX-bound HRP II structure most closely resembles a 3 10 -helix. Coincident with this structural change caused by Fe 3+ -PPIX binding, the formation of an intermolecular disulfide bond occurs between HRP II monomers. In vitro pull-down assays show an interaction between monomers that is dependent on the presence of Fe 3+ -PPIX. One model that best fits with the data reported here requires formation of 15 intermolecular bishistidyl ligated Fe 3+ -PPIX molecules arranged in a head to head fashion, which would then allow for the formation of an intermolecular disulfide bond. The structure best able to accommodate these requirements is a 3 10 -helix. Malaria, a disease caused by the Plasmodium parasite, continues to be one of the most important diseases in the world, resulting in 300-500 million infections and more than 1 million deaths every year, as estimated by the WHO (1). Of the Plasmodium parasites that cause malaria in humans, Plasmodium falciparum has the highest mortality rate. Inside the human host, P. falciparum eventually invades red blood cells where it rapidly grows and multiplies, producing multiple copies of new parasites within 48 h. To support this growth, the parasite ingests up to 75% of the host hemoglobin into the food vacuole, which is then digested by a number of proteases (2, 3). The resulting amino acids are then used as a source of nutrients by the parasite. In addition, the degradation of hemoglobin releases toxic free ferriprotoporphyrin IX (Fe 3+ -PPIX) 1 into the parasite food vacuole (4). Because of the toxic effect Fe 3+ -PPIX has on the parasite (5, 6), it is imperative for P. falciparum to detoxify free Fe 3+ -PPIX which it accomplishes in a unique way by forming hemozoin, an insoluble, crystalline form of Fe 3+ -PPIX. The structure of hemozoin involves the dimer- ization of Fe 3+ -PPIX molecules through reciprocal iron- carboxylate ligation. Hydrogen bonding occurs between the free carboxylates of separate dimers to form a chain of dimers (7). The need for a variety of effective antimalarials has increased dramatically because of resistance to the most common and inexpensive drug, chloroquine (8). Since humans do not rely on hemozoin formation for Fe 3+ -PPIX detoxification, this mechanism for Fe 3+ -PPIX disposal is an ideal target for antimalarial drug development. The exact mechanism for hemozoin formation by P. falciparum is still under debate (9), with reports demonstrating hemozoin formation through self-catalysis (10), in the presence of histidine-rich proteins (11) or lipids (12, 13), and through combinations of these factors (10, 13-15). The histidine-rich proteins are the only proteins to date that continue to be implicated in hemozoin formation. Studies by Choi et al. have shown that HRP II directly interacts with multiple Fe 3+ -PPIX molecules, increasing support for its role in Fe 3+ -PPIX detoxification (16). However, studies on parasites lacking HRP II and HRP III maintain the ability to produce hemozoin, indicating that these proteins, although capable, are not essential for hemozoin formation. This study at the same time does not rule out the participation of other histidine-rich proteins. Akompong et al. have recently investigated the subcellular localization of HRP II and found that 97% is exported to the erythrocyte cytosol with the remaining 3% located in the parasite food vacuole, suggesting that the main function of HRP II is not hemozoin formation (17). However, the population located in the food vacuole may still play an important role in hemozoin formation, while the cytosolic HRP II may be involved in Fe 3+ -PPIX scavanging. † This work was supported by the Burroughs-Wellcome Fund, New Initiatives in Malaria Research. * To whom correspondence should be addressed at the Department of Chemistry, University of California, Berkeley, 211 Lewis Hall, Berkeley, CA 94720-1460. Phone: (510) 643-9325. Fax: (510) 643- 9388. E-mail: marletta@berkeley.edu. ‡ Department of Chemistry, UCB. § Department of Molecular and Cell Biology, UCB. | Division of Physical Biosciences, LBNL. 1 Abbreviations: Fe 3+ -PPIX, ferric protoporphyrin IX; HRP II, histidine-rich protein II; HRP II(C274S), histidine-rich protein II Cys274 to Ser mutant; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CD, circular dichroism; PAGE, polyacrylamide gel electrophore- sis; MMTS, methyl methanethiosulfonate; DTT, dithiothreitol. 979 Biochemistry 2005, 44, 979-986 10.1021/bi048570p CCC: $30.25 © 2005 American Chemical Society Published on Web 12/24/2004