, 2003; Grüner et al., 2004; Cowman & Crabb, 2006; Iyer et al., 2007a, b). Little is known about how the large RH transmembrane proteins mediate their function during erythrocyte invasion, but a crucial step appears to be the proteolytic cleavage during the invasion process (Ogun & Holder, 1994; Triglia et al., 2009). Members of RH have been identified in all Plasmodium species analyzed so far, indicating the conserved function and importance of this protein family to the

malaria parasite (Iyer et al., 2007a; Rodriguez et al., 2008). In the rodent malaria parasite Plasmodium yoelii, selleck kinase inhibitor which has been widely used as a model to study host–parasite interactions (Landau & Gautret, 1998), the RH protein, termed Py235 (235 kDa in mass), has been shown to be a potential virulence factor that allows the parasite to invade a wider range of host erythrocytes (Freeman et al., 1980; Holder & Freeman, 1981). Py235 is also involved in the clonal phenotypic variation of merozoites (Preiser et al., 1999), enabling the parasite to evade immune responses and adapt to changes in PD-0332991 mouse the host environment during the invasion step (Snounou et al., 2000). Previously, a 94 kDa domain of Py235 of P. yoelii, which is highly conserved among the RBLs, has been found to selectively bind ATP and ADP, and is termed the nucleotide-binding domain (NBD94, Ramalingam et al., 2008). The amino acid sequence

483FNEIKEKLKHYNFDDFVKEE502 in NBD94 has been identified as a nucleotide-binding region as shown by photoaffinity labeling of the nucleotide analogue 8-N3-3′-biotinyl-ATP (Ramalingam

et al., 2008). The preference of MgATP over MgADP recognition is associated with specific structural alterations in the C-terminal domain of NBD94 as depicted by spectroscopic comparison of NBD94 and its C-terminal truncated form, NBD941–550, in which no nucleotide-dependent alteration could be observed (Ramalingam et al., 2008). This nucleotide effect in the recombinant protein is potentially significant, as demonstrated by a strong binding of Py235 to RBCs in the presence of MgATP, which becomes considerably reduced either in the presence of MgADP or in the absence of nucleotides oxyclozanide (Ramalingam et al., 2008). Based on these traits and the absence of significant ATPase activity of NBD94, this domain was suggested to serve as an ATP/ADP sensor during the invasion process (Ramalingam et al., 2008). More recently, the low-resolution solution and crystallographic structure of the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, have been determined, respectively (Grüber et al., 2010). The crystal structure of the hinge region, including residues 566–663, called NBD94566–663, consists of two helices 97.8 and 48.6 Å in length, linked by a loop. The region NBD94444–547 (residues 444–547 of NBD94) has been identified to form the nucleotide-binding segment, specific for ATP and ADP.