Xuecheng Zhang

Publications (7)20.71 Total impact

• Article: Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction.

  Xuecheng Zhang, Christopher G Adda, Andrew Low, Jiahai Zhang, Wen Zhang, Hongbin Sun, Xiaoming Tu, Robin F Anders, Raymond S Norton
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  ABSTRACT: Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.
  Biochemistry 02/2012; 51(7):1380-7. · 3.42 Impact Factor
• Article: Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima.

  Kelly Mai, Nicholas C Smith, Zhi-Ping Feng, Marilyn Katrib, Jan Slapeta, Iveta Slapetova, Michael G Wallach, Catherine Luxford, Michael J Davies, Xuecheng Zhang, Raymond S Norton, Sabina I Belli
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  ABSTRACT: Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles - wall-forming bodies - found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52-70%), with some α-helix (28-43%) but a relatively low percentage of β-sheet (1-11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein's propensity to form dityrosine bonds.
  International journal for parasitology 07/2011; 41(11):1157-64. · 3.39 Impact Factor
• Article: Identification of key residues involved in fibril formation by the conserved N-terminal region of Plasmodium falciparum merozoite surface protein 2 (MSP2).

  Xiaodong Yang, Christopher G Adda, Christopher A MacRaild, Andrew Low, Xuecheng Zhang, Weiguang Zeng, David C Jackson, Robin F Anders, Raymond S Norton
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  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.
  Biochimie 10/2010; 92(10):1287-95. · 3.02 Impact Factor
• Article: Solution conformation, backbone dynamics and lipid interactions of the intrinsically unstructured malaria surface protein MSP2.

  Xuecheng Zhang, Matthew A Perugini, Shenggen Yao, Christopher G Adda, Vincent J Murphy, Andrew Low, Robin F Anders, Raymond S Norton
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  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 alpha-helix or beta-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.
  Journal of Molecular Biology 06/2008; 379(1):105-21. · 4.00 Impact Factor
• Article: Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10 nAChR: structure and identification of key receptor-binding residues.

  Michael Ellison, Zhi-Ping Feng, Anthony J Park, Xuecheng Zhang, Baldomero M Olivera, J Michael McIntosh, Raymond S Norton
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  ABSTRACT: Alpha-conotoxins are small disulfide-constrained peptides from cone snails that act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide alpha-conotoxin RgIA (alpha-RgIA) is a member of the alpha-4,3 family of alpha-conotoxins and selectively blocks the alpha9alpha10 nAChR subtype, in contrast to another well-characterized member of this family, alpha-conotoxin ImI (alpha-ImI), which is a potent inhibitor of the alpha7 and alpha3beta2 nAChR subtypes. In this study, we have altered side chains in both the four-residue and the three-residue loops of alpha-RgIA, and have modified its C-terminus. The effects of these changes on activity against alpha9alpha10 and alpha7 nAChRs were measured; the solution structures of alpha-RgIA and its Y10W, D5E, and P6V analogues were determined from NMR data; and resonance assignments were made for alpha-RgIA [R9A]. The structures for alpha-RgIA and its three analogues were well defined, except at the chain termini. Comparison of these structures with reported structures of alpha-ImI reveals a common two-loop backbone architecture within the alpha-4,3 family, but with variations in side-chain solvent accessibility and orientation. Asp5, Pro6, and Arg7 in loop 1 are critical for blockade of both the alpha9alpha10 and the alpha7 subtypes. In loop 2, alpha-RgIA [Y10W] had activity near that of wild-type alpha-RgIA, with high potency for alpha9alpha10 and low potency for alpha7, and had a structure similar to that of wild type. By contrast, Arg9 in loop 2 is critical for specific binding to the alpha9alpha10 subtype, probably because it is larger and more solvent accessible than Ala9 in alpha-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the alpha9alpha10 nAChR subtype, which is a target for the development of analgesics for the treatment of chronic neuropathic pain.
  Journal of Molecular Biology 05/2008; 377(4):1216-27. · 4.00 Impact Factor
• Article: Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain.

  Andrew Low, Indu R Chandrashekaran, Christopher G Adda, Shenggen Yao, Jennifer K Sabo, Xuecheng Zhang, Alfreda Soetopo, Robin F Anders, Raymond S Norton
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  ABSTRACT: Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.
  Biopolymers 10/2007; 87(1):12-22. · 2.87 Impact Factor
• Article: α-RgIA, a Novel Conotoxin That Blocks the α9α10 nAChR: Structure and Identification of Key Receptor-Binding Residues

  Michael Ellison, Zhi-Ping Feng, Anthony J. Park, Xuecheng Zhang, Baldomero M. Olivera, J. Michael McIntosh, Raymond S. Norton
  [show abstract] [hide abstract]
  ABSTRACT: α-Conotoxins are small disulfide-constrained peptides from cone snails that act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide α-conotoxin RgIA (α-RgIA) is a member of the α-4,3 family of α-conotoxins and selectively blocks the α9α10 nAChR subtype, in contrast to another well-characterized member of this family, α-conotoxin ImI (α-ImI), which is a potent inhibitor of the α7 and α3β2 nAChR subtypes. In this study, we have altered side chains in both the four-residue and the three-residue loops of α-RgIA, and have modified its C-terminus. The effects of these changes on activity against α9α10 and α7 nAChRs were measured; the solution structures of α-RgIA and its Y10W, D5E, and P6V analogues were determined from NMR data; and resonance assignments were made for α-RgIA [R9A]. The structures for α-RgIA and its three analogues were well defined, except at the chain termini. Comparison of these structures with reported structures of α-ImI reveals a common two-loop backbone architecture within the α-4,3 family, but with variations in side-chain solvent accessibility and orientation. Asp5, Pro6, and Arg7 in loop 1 are critical for blockade of both the α9α10 and the α7 subtypes. In loop 2, α-RgIA [Y10W] had activity near that of wild-type α-RgIA, with high potency for α9α10 and low potency for α7, and had a structure similar to that of wild type. By contrast, Arg9 in loop 2 is critical for specific binding to the α9α10 subtype, probably because it is larger and more solvent accessible than Ala9 in α-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the α9α10 nAChR subtype, which is a target for the development of analgesics for the treatment of chronic neuropathic pain.
  Journal of Molecular Biology.