Identification of key residues involved in fibril formation by the conserved N-terminal region of Plasmodium falciparum merozoite surface protein 2 (MSP2)

Abstract

Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. MSP2 is assumed to have a role in erythrocyte invasion and is a leading vaccine candidate. Recombinant MSP2 forms amyloid-like fibrils upon storage, as do peptides corresponding to sequences in the conserved N-terminal region, which constitutes the structural core of fibrils formed by full-length MSP2. We have investigated the roles of individual residues in fibril formation and local ordered structure in two peptides, a recombinant 25-residue peptide corresponding to the entire N-terminal domain of mature MSP2 and an 8-residue peptide from the central region of this domain (residues 8-15). Both peptides formed fibrils that were similar to amyloid-like fibrils formed by full-length MSP2. Phe11 and Ile12 have important roles both in stabilising local structure in these peptides and promoting fibril formation; the F11A and I12A mutants of MSP2(8-15) were essentially unstructured in solution and fibril formation at pH 7.4 and 4.7 was markedly retarded. The T10A mutant showed intermediate behaviour, having a less well defined structure than wild-type and slower fibril formation at pH 7.4. The mutation of Phe11 and Ile12 in MSP2(1-25) significantly retarded but did not abolish fibril formation, indicating that these residues also play a key role in fibril formation by the entire N-terminal conserved region. These mutations had little effect on the aggregation of full-length MSP2, however, suggesting that regions outside the conserved N-terminus have unanticipated importance for fibril formation in the full-length protein.

Figure 1

Sequences of peptides used in this study and the relationships between them. S: 19-residue signal peptide which is predicted to be cleaved to yield the mature protein. N: 25-residue N-terminal region conserved between allelic families. Variable region: Central repeat region that contains the highly polymorphic region of MSP2. C: Conserved 74-residue C-terminal domain with a disulfide bond. A: GPI Anchor. The MSP2_1–25 and MSP2_8–15 (boxed) peptides shown formed the basis of this study. All numbering of residues in this study is relative to mature full-length MSP2.

Figure 2

Fibril formation by wild-type MSP2_8–15 (●) and MSP2_1–25 (◽). The peptides were heated at 80 °C and reactions were established with 100 μM of peptide in PBS at pH 7.4 and incubated at room temperature. Fibril formation was monitored by measuring ThT fluorescence.

Figure 3

Rates of fibril formation of wild-type MSP2_8–15 and seven mutants. (A) Reactions were established by diluting the peptide to 100 μM in PBS, pH 7.4. At various time points, samples were diluted into PBS containing 30 μM ThT and fibril formation was monitored by measuring ThT fluorescence and plotted against time. (B) Relative methyl ¹H NMR signal intensities as a function of time for MSP2_8–15 and mutants.

Figure 4

Electron micrographs of the fibrils formed by (A) MSP2_8–15 N9A, (B) MSP2_8–15 F11A and (C) MSP2_8–15 I12A. Samples were taken from the NMR tubes (cf. Figure 3B), negatively stained and viewed using transmission EM. The scale bar represents 100 nm.

Figure 5

Kinetics of fibril formation by wild-type MSP2_1–25 (◽), [Y7A,Y16A]MSP2_1–25.(●) and [F11A,I12A]MSP2_1–25. (▲). In 96 well plates, quadruplicate reactions were established by diluting the peptide to 100 μM in PBS containing 30 μM ThT and fibril formation was monitored by measuring ThT fluorescence. The average values for each protein were expressed as a fraction of the maximum value and plotted against time.

Figure 6

Backbone chemical shift differences from wild-type for MSP2_8–15 mutants at 5 °C. (A) S8A (B) N9A (C) T10A (D) F11A (E) I12A (F) N13A (G) N14A. Chemical shifts values of wild-type and mutant peptides were corrected for the differences between random coil values before subtracting wild-type values from mutant values.

Figure 7

NOE restraints numbers for MSP2_8–15 mutant peptides at 5 °C. NOE numbers are obtained from NOESY spectra (mixing time 250 ms; 600 MHz) recorded at peptide concentration of 5 mg/mL in 10 mM sodium acetate at pH 4.7.

Figure 8

Families of structures for wild-type MSP2_8–15, and T10A, F11A and I12A mutants calculated using CYANA at same orientation. Structures were superimposed over the backbone heavy atoms (N, C^α and C) of residues Asn9 to Asn14.

Figure 9

Kinetics of fibril formation of full-length MSP2 mutants. (A) Reactions were established with 40 μM monomeric MSP2 for each of wt FC27 MSP2 (●), F11A FC27 MSP2 (○), I12A FC27 MSP2 (◽), and [F11A,I12A] FC27 MSP2 (◊) in PBS containing 30 μM ThT. The measured ThT fluorescence intensity was plotted against time. (B) Following the establishment of the fibril forming reactions with 160 uM of wt 3D7 MSP2 (●), F11A 3D7 MSP2 (○), I12A 3D7 MSP2 (◽), and [F11A,I12A] 3D7 MSP2 (◊), SEC was used to determine the amount of monomeric MSP2 remaining, represented by the area under the peak corresponding to monomeric MSP2, and was plotted for each time interval.

Figure 10

Electron micrographs of the fibrils formed by (A) wt 3D7 MSP2, (B) F11A 3D7 MSP2 (C) I12A 3D7 MSP2 and (D) [F11A,I12A] 3D7 MSP2. Peptide conditions are same as those in Figure 9B. Samples were negatively stained and viewed using transmission EM. The scale bar represents 500 nm.