Dissection of Merozoite Surface Protein 3, a Representative of a Family of Plasmodium falciparum Surface Proteins, Reveals an Oligomeric and Highly Elongated Molecule

Abstract

Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the fulllength molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of ␣-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The fulllength MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 M. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f 0 > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.Plasmodium falciparum, the parasitic agent that causes most cases of fatal malaria, is estimated to infect over 500 million people annually (1), causing 1-3 million deaths among young children in sub-Saharan Africa. The rise of parasite resistance to currently available drugs has made malaria treatment difficult. Vaccines have great potential as lower cost alternatives to widespread drug treatment, since they may provide long lived protection from disease. The P. falciparum blood stage surface protein MSP3 2 (2, 3) is a vaccine candidate, since it is known to be a target of the immune response. Immunizations with several forms of recombinant MSP3 were each shown to protect monkeys against parasite challenge (4, 5). MSP3-specific antibodies in the presence of monocytes mediated killing of parasites in an antibody-dependent cellular inhibition assay (6). Homologs to MSP3 have been identified in several Plasmodium species (7-9), indicating that the molecule plays a conserved role in blood stage parasites. The P. falciparum MSP3 sequence predicts several domains ( Fig. 1): a central domain that includes three blocks of imperfect Ala heptad repeats of the sequence pattern Ala-X-X-Ala-X-X-X, a second central region rich in Glu residues, and a C-terminal leucine zipper-like domain (2, 3). Secondary structure algorithms predict MSP3 to be largely ␣-helical, especially in the Ala heptad repeat region that is pre...

Insights into Duffy Binding-like Domains through the Crystal Structure and Function of the Merozoite Surface Protein MSPDBL2 from Plasmodium falciparum

“…Coloring of the electrostatic potential is from red (Ϫ15 kJ mol Ϫ1 ), through white (0 kJ mol it remains possible that dimerization is still required for ligand recognition. In the full-length protein, dimerization is probably facilitated by the C-terminal SPAM domain present in both PfMSPDBL1 and -2, as is the case for MSP3 (46). Dimerization via this motif would likely orient two DBL domains in a parallel fashion; however, flexibility within the linker region between the DBL and SPAM domains may allow for a variety of DBL dimer interfaces.…”

“…The leucine zipper-like region has been shown to be responsible for oligomerization of MSP3. Analysis of the physical properties of MSP3 has suggested that it forms a highly extended and oligomeric structure on the merozoite surface that would allow it to interact with the red blood cell at relatively long distances (46), most notably, dimerization of MPS3 is solely through the leucine zipper-like domain. As PfMSPDBL1 and -2 bind red blood cells through the DBL domains and share with MSP3 a SPAM domain at the C terminus, they are also likely to be long extended molecules able to bind to their specific receptors.…”

Plasmodium falciparum Merozoite Surface Protein 3

“…A vaccine based on MSP3 N-terminal fragment is already in human trials (9,10). In addition to being a potential vaccine candidate, two other characteristics of MSP3 stand out as follows: first, MSP3, although a soluble protein, forms oligomers, and second, it can bind to heme, although the significance of these characteristics is not well understood (7,11).…”

“…Other surface proteins, including MSP3, MSP6, MSP7, and MSP9 (also known as ABRA), are soluble proteins and are present on the merozoite surface as a protein complex, possibly through protein-protein interactions (4). Of these proteins, MSP3 is of particular interest, both as a vaccine candidate antigen as well as for its peculiar structural characteristics (5)(6)(7).…”

Interactions between Merozoite Surface Proteins 1, 6, and 7 of the Malaria ParasitePlasmodium falciparum

“…MSP-6 is related to MSP-3 (3) based on a high sequence similarity at the C terminus, which can give rise to antibodies reacting with both proteins (10,11). Recently, it has been shown that MSP-3 can form highly elongated dimers and tetramers via its C terminus containing a predicted coiled-coil region (12).…”