Solution Conformation, Backbone Dynamics and Lipid Interactions of the Intrinsically Unstructured Malaria Surface Protein MSP2

Abstract

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little α-helix or β-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.

Introduction

Malaria is one of the most important infectious diseases in the world, resulting in half a billion clinical episodes and as many as two million deaths annually.1, 2, 3 Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the merozoite surface of Plasmodium falciparum, has been shown to be a promising vaccine target.4, 5 MSP2 is a glycosyl-phosphatidyl-inositol (GPI)-anchored protein with conserved N- and C-terminal domains, and a variable central region (Fig. 1). The patterns of diversity in this central region have led to all MSP2 alleles being classified into two families, FC27 and 3D7 (Fig. 1).6, 7, 8, 9, 10, 11 Recent studies have shown that full-length MSP2 is an intrinsically unstructured protein (IUP) and prone to form amyloid-like fibrils, in which the N-terminal region (MSP2_1–25) has a key role.12, 13, 14 In addition, there is evidence that MSP2 exists in an oligomeric form on the merozoite surface.¹²

Intrinsically unstructured proteins are widespread,15, 16, 17 and the proteome of the malaria parasite is predicted to contain a remarkably high proportion of this class of proteins.¹⁸ Some IUPs are involved in important regulatory functions and may fold into ordered structures only upon recognition of their biological partners, while others are prone to form amyloid fibrils that are associated with neurodegenerative conditions and systemic or localized amyloidoses.¹⁹ Recently, some IUPs were found to form functional amyloid-like fibrils on bacterial and fungal surfaces where they may facilitate attachment to host cells or other surfaces.19, 20

Understanding the structure and dynamics of the unstructured state of IUPs can provide valuable insight into the conformational space available to these proteins and ultimately to their function.21, 22 This class of proteins cannot be described adequately by a single conformation, but rather by an ensemble of rapidly interconverting conformations.²³ Indeed, IUPs are unsuitable for study by X-ray crystallography because of their disordered nature, but high-resolution NMR can provide detailed insights into their structure and dynamics, and studies of many such proteins have been reported.24, 25, 26, 27, 28, 29, 30 Experimental challenges associated with NMR studies of disordered proteins include extensive peak overlap, particularly for proton resonances, and their tendency to aggregate in solution. On the other hand, the substantial intrinsic dispersion of ¹⁵N and ¹³CO chemical shifts, the fact that chemical shifts tend to be close to random coil values, and the typically sharp peaks in the spectra of disordered proteins all favor resonance assignments, which are crucial for detailed NMR studies of proteins.²⁴

The aims of this study were to characterize the structure and stability of recombinant MSP2 in aqueous solution by employing circular dichroism (CD) spectroscopy, analytical ultracentrifugation and NMR spectroscopy. Our results show that MSP2 is largely unfolded, but with some nascent structures present under all the conditions investigated. Residue-specific NMR studies on the FC27 isoform of MSP2 defined three motionally restricted regions in the protein. We further identified a key role of the N-terminal helical region in the interaction of FC27 MSP2 with lipid micelles. The results provide a clear picture of the solution properties of monomeric FC27 MSP2, and provide a basis for understanding the nature of MSP2 on the merozoite surface.