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The Structure of Plasmodium falciparum Blood-Stage 6-Cys Protein Pf41 Reveals an Unexpected Intra-Domain Insertion Required for Pf12 Coordination.

Parker ML, Peng F, Boulanger MJ - PLoS ONE (2015)

Bottom Line: Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1.Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12.Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada.

ABSTRACT
Plasmodium falciparum is an apicomplexan parasite and the etiological agent of severe human malaria. The complex P. falciparum life cycle is supported by a diverse repertoire of surface proteins including the family of 6-Cys s48/45 antigens. Of these, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with the glycophosphatidylinositol-anchored Pf12. Our recent structural characterization of Pf12 revealed two juxtaposed 6-Cys domains (D1 and D2). Pf41, however, contains an additional segment of 120 residues predicted to form a large spacer separating its two 6-Cys domains. To gain insight into the assembly mechanism and overall architecture of the Pf12-Pf41 complex, we first determined the 2.45 Å resolution crystal structure of Pf41 using zinc single-wavelength anomalous dispersion. Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1. Notably, the ID is largely proteolyzed in the final structure suggesting inherent flexibility. To assess the contribution of the ID to complex formation, we engineered a form of Pf41 where the ID was replaced by a short glycine-serine linker and showed by isothermal titration calorimetry that binding to Pf12 was abrogated. Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12. Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.

No MeSH data available.


Related in: MedlinePlus

The structure of Pf41 reveals that a large sequence insertion predominately maps between two β-strands in D1.(A) Schematic comparison of predicted Pf41 domain organization with established Pf12 tandem 6-Cys domains. SP, signal peptide; D1, domain 1; D2, domain 2; arrow indicates GPI anchor attachment site. Black bars indicate disulfide connectivity. (B) Anomalous difference electron density map of Pf41 calculated at 8 sigma (orange mesh) around one of two high confidence zinc sites (grey sphere) used for phasing. Two Glu residues (Glu 132 and 134) were found to coordinate the zinc ion (along with His23 and Glu369 of two neighboring chains, not shown) and are shown in green ball-and-stick colored by element. (C) Left/middle–Orthogonal views of the Pf41 structure, shown as a cartoon backbone colored as in (A). Disulfides are shown in the left panel as ball-and-stick colored by element. A four residue un-modeled region in D1 is indicated by a dotted green connecting loop. Grey dotted lines extending out of α1” (inserted domain-like region, ID) and into β9 (D1) indicate the un-modeled region of the ID (~85 residues). Right–green surface of Pf41 in same orientation as (C, middle) colored as in (A), with the previously undefined region shown with a semi-transparent grey surface with underlying cartoon. (D) Left–Lys349 shown as green sticks colored by element with the positive Fo-Fc map shown as an orange mesh contoured at 2.5σ. Right–final σ-A weighted 2Fo-Fc electron density map shown as a purple mesh contoured at 1σ around the dimethylated lysines (MLY347 and 349). The symmetry mate against which the methylated lysines pack is shown as a semi-transparent grey surface. (E) Sequence of the ID and flanking secondary structure elements colored as in (A). Regions observed in the structure have a solid underline; un-modelled sequence and predicted secondary structure elements are indicated by dashed lines.
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pone.0139407.g001: The structure of Pf41 reveals that a large sequence insertion predominately maps between two β-strands in D1.(A) Schematic comparison of predicted Pf41 domain organization with established Pf12 tandem 6-Cys domains. SP, signal peptide; D1, domain 1; D2, domain 2; arrow indicates GPI anchor attachment site. Black bars indicate disulfide connectivity. (B) Anomalous difference electron density map of Pf41 calculated at 8 sigma (orange mesh) around one of two high confidence zinc sites (grey sphere) used for phasing. Two Glu residues (Glu 132 and 134) were found to coordinate the zinc ion (along with His23 and Glu369 of two neighboring chains, not shown) and are shown in green ball-and-stick colored by element. (C) Left/middle–Orthogonal views of the Pf41 structure, shown as a cartoon backbone colored as in (A). Disulfides are shown in the left panel as ball-and-stick colored by element. A four residue un-modeled region in D1 is indicated by a dotted green connecting loop. Grey dotted lines extending out of α1” (inserted domain-like region, ID) and into β9 (D1) indicate the un-modeled region of the ID (~85 residues). Right–green surface of Pf41 in same orientation as (C, middle) colored as in (A), with the previously undefined region shown with a semi-transparent grey surface with underlying cartoon. (D) Left–Lys349 shown as green sticks colored by element with the positive Fo-Fc map shown as an orange mesh contoured at 2.5σ. Right–final σ-A weighted 2Fo-Fc electron density map shown as a purple mesh contoured at 1σ around the dimethylated lysines (MLY347 and 349). The symmetry mate against which the methylated lysines pack is shown as a semi-transparent grey surface. (E) Sequence of the ID and flanking secondary structure elements colored as in (A). Regions observed in the structure have a solid underline; un-modelled sequence and predicted secondary structure elements are indicated by dashed lines.

Mentions: Recent structural analysis of Pf12 provided the first insight into the architecture of the 6-Cys domain and the organization of the tandem repeats with the two disulfide pinned β-sandwich domains connected via a short linker [5, 15]. Intriguingly, Pf41 incorporates an additional 120 residues of unknown structure that appear to form a spacer linking the two predicted 6-Cys domains (D1 and D2) (Fig 1A). Based on the expected domain organization of Pf41 [17], the structure of Pf12 [15], and chemical cross-linking data [15], we previously proposed a preliminary model where Pf12 and Pf41 adopt an antiparallel organization [15]. A weakness of this model, however, is the absence of structural information describing Pf41 and, in particular, the large sequence insertion. To address this limitation, we report here the crystal structure of Pf41 and complement the structural data with solution binding studies to reveal the molecular determinants of Pf12-Pf41 complex formation. Based on these data, we re-evaluate and refine the model of the assembly mechanism and overall architecture of the Pf12-Pf41 heterodimer.


The Structure of Plasmodium falciparum Blood-Stage 6-Cys Protein Pf41 Reveals an Unexpected Intra-Domain Insertion Required for Pf12 Coordination.

Parker ML, Peng F, Boulanger MJ - PLoS ONE (2015)

The structure of Pf41 reveals that a large sequence insertion predominately maps between two β-strands in D1.(A) Schematic comparison of predicted Pf41 domain organization with established Pf12 tandem 6-Cys domains. SP, signal peptide; D1, domain 1; D2, domain 2; arrow indicates GPI anchor attachment site. Black bars indicate disulfide connectivity. (B) Anomalous difference electron density map of Pf41 calculated at 8 sigma (orange mesh) around one of two high confidence zinc sites (grey sphere) used for phasing. Two Glu residues (Glu 132 and 134) were found to coordinate the zinc ion (along with His23 and Glu369 of two neighboring chains, not shown) and are shown in green ball-and-stick colored by element. (C) Left/middle–Orthogonal views of the Pf41 structure, shown as a cartoon backbone colored as in (A). Disulfides are shown in the left panel as ball-and-stick colored by element. A four residue un-modeled region in D1 is indicated by a dotted green connecting loop. Grey dotted lines extending out of α1” (inserted domain-like region, ID) and into β9 (D1) indicate the un-modeled region of the ID (~85 residues). Right–green surface of Pf41 in same orientation as (C, middle) colored as in (A), with the previously undefined region shown with a semi-transparent grey surface with underlying cartoon. (D) Left–Lys349 shown as green sticks colored by element with the positive Fo-Fc map shown as an orange mesh contoured at 2.5σ. Right–final σ-A weighted 2Fo-Fc electron density map shown as a purple mesh contoured at 1σ around the dimethylated lysines (MLY347 and 349). The symmetry mate against which the methylated lysines pack is shown as a semi-transparent grey surface. (E) Sequence of the ID and flanking secondary structure elements colored as in (A). Regions observed in the structure have a solid underline; un-modelled sequence and predicted secondary structure elements are indicated by dashed lines.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587554&req=5

pone.0139407.g001: The structure of Pf41 reveals that a large sequence insertion predominately maps between two β-strands in D1.(A) Schematic comparison of predicted Pf41 domain organization with established Pf12 tandem 6-Cys domains. SP, signal peptide; D1, domain 1; D2, domain 2; arrow indicates GPI anchor attachment site. Black bars indicate disulfide connectivity. (B) Anomalous difference electron density map of Pf41 calculated at 8 sigma (orange mesh) around one of two high confidence zinc sites (grey sphere) used for phasing. Two Glu residues (Glu 132 and 134) were found to coordinate the zinc ion (along with His23 and Glu369 of two neighboring chains, not shown) and are shown in green ball-and-stick colored by element. (C) Left/middle–Orthogonal views of the Pf41 structure, shown as a cartoon backbone colored as in (A). Disulfides are shown in the left panel as ball-and-stick colored by element. A four residue un-modeled region in D1 is indicated by a dotted green connecting loop. Grey dotted lines extending out of α1” (inserted domain-like region, ID) and into β9 (D1) indicate the un-modeled region of the ID (~85 residues). Right–green surface of Pf41 in same orientation as (C, middle) colored as in (A), with the previously undefined region shown with a semi-transparent grey surface with underlying cartoon. (D) Left–Lys349 shown as green sticks colored by element with the positive Fo-Fc map shown as an orange mesh contoured at 2.5σ. Right–final σ-A weighted 2Fo-Fc electron density map shown as a purple mesh contoured at 1σ around the dimethylated lysines (MLY347 and 349). The symmetry mate against which the methylated lysines pack is shown as a semi-transparent grey surface. (E) Sequence of the ID and flanking secondary structure elements colored as in (A). Regions observed in the structure have a solid underline; un-modelled sequence and predicted secondary structure elements are indicated by dashed lines.
Mentions: Recent structural analysis of Pf12 provided the first insight into the architecture of the 6-Cys domain and the organization of the tandem repeats with the two disulfide pinned β-sandwich domains connected via a short linker [5, 15]. Intriguingly, Pf41 incorporates an additional 120 residues of unknown structure that appear to form a spacer linking the two predicted 6-Cys domains (D1 and D2) (Fig 1A). Based on the expected domain organization of Pf41 [17], the structure of Pf12 [15], and chemical cross-linking data [15], we previously proposed a preliminary model where Pf12 and Pf41 adopt an antiparallel organization [15]. A weakness of this model, however, is the absence of structural information describing Pf41 and, in particular, the large sequence insertion. To address this limitation, we report here the crystal structure of Pf41 and complement the structural data with solution binding studies to reveal the molecular determinants of Pf12-Pf41 complex formation. Based on these data, we re-evaluate and refine the model of the assembly mechanism and overall architecture of the Pf12-Pf41 heterodimer.

Bottom Line: Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1.Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12.Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada.

ABSTRACT
Plasmodium falciparum is an apicomplexan parasite and the etiological agent of severe human malaria. The complex P. falciparum life cycle is supported by a diverse repertoire of surface proteins including the family of 6-Cys s48/45 antigens. Of these, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with the glycophosphatidylinositol-anchored Pf12. Our recent structural characterization of Pf12 revealed two juxtaposed 6-Cys domains (D1 and D2). Pf41, however, contains an additional segment of 120 residues predicted to form a large spacer separating its two 6-Cys domains. To gain insight into the assembly mechanism and overall architecture of the Pf12-Pf41 complex, we first determined the 2.45 Å resolution crystal structure of Pf41 using zinc single-wavelength anomalous dispersion. Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1. Notably, the ID is largely proteolyzed in the final structure suggesting inherent flexibility. To assess the contribution of the ID to complex formation, we engineered a form of Pf41 where the ID was replaced by a short glycine-serine linker and showed by isothermal titration calorimetry that binding to Pf12 was abrogated. Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12. Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.

No MeSH data available.


Related in: MedlinePlus