Solution structure of an EGF module pair from the Plasmodium falciparum merozoite surface protein 1¹

Abstract

The solution structure of the 96-residue C-terminal fragment of the merozoite surface protein 1 (MSP-1) from Plasmodium falciparum has been determined using nuclear magnetic resonance (NMR) spectroscopic measurements on uniformly ¹³C/¹⁵N-labelled protein, efficiently 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) membrane anchor site, which is consistent with the involvement of a membrane-bound proteinase in the processing of MSP-1 during erythrocyte invasion. This structure, which is the first protozoan EGF example to be determined, contrasts with the elongated structures seen for EGF-module pairs having shared Ca²⁺-ligation sites at their interface, as found, for example, in fibrillin-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 structure by considering specific MSP-1 mutants.

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 parasite and its interaction with the host. One approach focuses on attempts to understand fundamental aspects of parasite invasion of erythrocytes, a critical 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 merozoite surface there is a glycosyl phosphatidyl inositol-anchored protein complex derived from a ∼200 kDa precursor (merozoite surface protein 1 (MSP-1)) and associated polypeptides. The precursor is first 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 currently the leading candidate for development of a vaccine against the blood stages of the malaria parasite Diggs et al 1993, Stoute and 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 residue sequence with a characteristic disulphide bonding pattern, and such domains occur frequently in extracellular modular proteins of animals. In the MSP-1 C-terminal fragment each of the motifs contains six Cys residues that have been proposed to form three disulphide bonds and each motif has a partial match to the EGF consensus (see Figure 1). However, because the degree of similarity is limited and since the pattern of its disulphide bonding is not known, the designation of the MSP-1 C-terminal fragment as being composed of EGF-like structures has been regarded as tentative. Other relatively divergent potential EGF-like sequences occur in Plasmodium, but previous structure determinations have been confined to those from metazoan organisms (Campbell & Downing, 1998). It is important to determine the three-dimensional structure of the MSP-1 C-terminal fragment, since this could assist in the design of candidate vaccines and help in understanding antibody interactions with this fragment. Some monoclonal antibodies (mAbs) that bind to this fragment inhibit the proteolytic cleavage and erythrocyte invasion, suggesting that cleavage is a prerequisite for invasion (Blackman et al., 1994). Other mAbs that bind to the MSP-1 C-terminal fragment do not inhibit processing or invasion but block the binding of the inhibitory neutralizing antibodies. In the presence of blocking antibodies, inhibitory antibodies are ineffective and invasion proceeds. The balance between inhibitory and blocking antibodies induced by immunization may be a critical factor in determining whether or not the immune response is effective in preventing invasion (Guevara Patiño et al., 1997).

As a step towards understanding the interactions of antibodies with the MSP-1 C-terminal antigen, we have determined its solution structure, including the pattern of disulphide bonding, using NMR techniques.