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

Brandt R Burgess,David N Garboczi,Peter Schuck

doi:10.1074/jbc.m506753200

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 full-length molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of alpha-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The full-length 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 mum. 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.

Highlights

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 P. falciparum blood stage surface protein MSP32 [2, 3] is a vaccine candidate, since it is known to be a target of the immune response
A similar interaction between the C-terminal region of merozoite surface protein 3 (MSP3) and another surface protein may be how MSP3 associates with the merozoite surface

Summary

Introduction

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 P. falciparum blood stage surface protein MSP32 [2, 3] is a vaccine candidate, since it is known to be a target of the immune response. 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]. The homologous P. falciparum merozoite surface proteins MSP6 [11, 12] and MSP11 [13] contain Glu-rich and C-terminal leucine zipper-like domains and, together with MSP3, may identify a new family of proteins. A similar interaction between the C-terminal region of MSP3 and another surface protein may be how MSP3 associates with the merozoite surface. Our results show that 1) the full-length recombinant MSP3 has high percentages of ␣-helix and random coil; 2) full-length MSP3 forms both dimers and tetramers in solution, with the tetramer apparently formed by an “end-to-end” arrangement of two dimers; 3) the oligomerization domain resides in the 60 residues at the C terminus of the full-length protein; 4) the MSP3 dimer and tetramer are highly elongated molecules

Methods

Results

Conclusion