Solution Structure of an EGF Module Pair from the Plasmodium falciparum Merozoite Surface Protein 1 William D. Morgan 1 , Berry Birdsall 1 , Thomas A. Frenkiel 2 , Michael G. Gradwell 2 , Petra A. Burghaus 3 , Shabih E. H. Syed 3 , Chairat Uthaipibull 3 , Anthony A. Holder 3 and James Feeney 1 * 1 Molecular Structure Division 2 Biomedical NMR Centre 3 Parasitology Division National Institute for Medical Research The Ridgeway Mill Hill, London, NW7 1AA UK The solution structure of the 96-residue C-terminal fragment of the mero- zoite surface protein 1 (MSP-1) from Plasmodium falciparum has been determined using nuclear magnetic resonance (NMR) spectroscopic measurements on uniformly 13 C/ 15 N-labelled protein, ef®ciently expressed in the methylotrophic yeast Komagataella (Pichia) pastoris. The structure has two domains with epidermal growth factor (EGF)-like folds with a novel domain interface for the EGF domain pair interactions, formed from a cluster of hydrophobic residues. This gives the protein a U-shaped overall structure with the N-terminal proteolytic processing site close to the C-terminal glycosyl phosphatidyl inositol (GPI) mem- brane anchor site, which is consistent with the involvement of a mem- brane-bound proteinase in the processing of MSP-1 during erythrocyte invasion. This structure, which is the ®rst protozoan EGF example to be determined, contrasts with the elongated structures seen for EGF-module pairs having shared Ca 2 -ligation sites at their interface, as found, for example, in ®brillin-1. Recognition surfaces for antibodies that inhibit processing and invasion, and antibodies that block the binding of these inhibitory antibodies, have been mapped on the three-dimensional struc- ture by considering speci®c MSP-1 mutants. # 1999 Academic Press Keywords: protein structure; P. falciparum; merozoite surface protein; MSP-1; EGF modules *Corresponding author Introduction Malaria is the most serious vector-borne human disease and there is an urgent need for a more detailed understanding of the biology of the para- site and its interaction with the host. One approach focuses on attempts to understand fundamental aspects of parasite invasion of erythrocytes, a criti- cal step in the cyclic asexual blood stage of the parasite life cycle. In this process, the merozoite form of the human malaria parasite, Plasmodium falciparum, attaches to and invades an erythrocyte and then undergoes intracellular multiplication prior to release of further merozoites. On the mero- zoite surface there is a glycosyl phosphatidyl inosi- tol-anchored protein complex derived from a 200 kDa precursor (merozoite surface protein 1 (MSP-1)) and associated polypeptides. The precur- sor is ®rst cleaved into four pieces in a primary processing step that occurs on merozoite release; then, at or just before invasion most of the MSP-1 complex is shed from the surface by proteolytic cleavage (secondary processing), leaving only a 96 amino acid residue C-terminal fragment bound to the surface of the invading parasite (Blackman et al., 1990). This MSP-1 C-terminal fragment is cur- rently the leading candidate for development of a vaccine against the blood stages of the malaria parasite (Diggs et al., 1993; Stoute & Ballou, 1998). On the basis of amino acid sequence similarities, it has been suggested that this fragment is composed of two epidermal growth factor (EGF)-like motifs (see sequence in Figure 1) (Blackman et al., 1991). An EGF-like motif consists of a 45-50 amino acid E-mail address of the corresponding author: jfeeney@nimr.mrc.ac.uk Abbreviations used: MSP-1, merozoite surface protein 1; EGF, epidermal growth factor; mAb, monoclonal antibody; HSQC (HMQC), heteronuclear single (multiple) quantum coherence spectroscopy; NOE (NOESY), nuclear Overhauser effect (spectroscopy); ROE (ROESY), rotating frame nuclear Overhauser effect (spectroscopy). Article No. jmbi.1999.2753 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 289, 113±122 0022-2836/99/210113±10 $30.00/0 # 1999 Academic Press