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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

A refined Pf12-Pf41 heterodimeric assembly model.(A) Cross-linking designations revised from [15]; Pf41 ID specific interactions are bolded and shaded light grey. (B) Refined model of Pf12-Pf41 complex generated by manual docking and guided by cross-linking data. Pf12 is shown as a magenta surface, and Pf41 as a green surface except a model of the ID generated in iTASSER [42] displayed as a grey semi-transparent surface. The Pf41 ID model represents the relative size of this region compared to D1 and D2 and approximates how the ID could contact both Pf12 D1 and D2, but further studies probing the detailed structure and flexibility of this region are still needed. A previously identified Pf41 ID phosphorylation site [43] is shown in yellow and indicated by a yellow arrow. The black dotted line indicates uncertainty in the exact interface between the tandem 6-Cys domains of Pf12 and Pf41.
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pone.0139407.g005: A refined Pf12-Pf41 heterodimeric assembly model.(A) Cross-linking designations revised from [15]; Pf41 ID specific interactions are bolded and shaded light grey. (B) Refined model of Pf12-Pf41 complex generated by manual docking and guided by cross-linking data. Pf12 is shown as a magenta surface, and Pf41 as a green surface except a model of the ID generated in iTASSER [42] displayed as a grey semi-transparent surface. The Pf41 ID model represents the relative size of this region compared to D1 and D2 and approximates how the ID could contact both Pf12 D1 and D2, but further studies probing the detailed structure and flexibility of this region are still needed. A previously identified Pf41 ID phosphorylation site [43] is shown in yellow and indicated by a yellow arrow. The black dotted line indicates uncertainty in the exact interface between the tandem 6-Cys domains of Pf12 and Pf41.

Mentions: The structural and solution binding data reported here provided an opportunity to re-evaluate our previous Pf12-Pf41 cross-linking data [15]. Originally, cross-linked peptides were assigned to Pf41 D1, Linker, or D2 based on the previously predicted architecture of Pf41 with the spacer/linker region separating the two 6-Cys domains (Fig 1A). However, with the unambiguous domain assignments enabled by our Pf41 structure (Fig 3A), we can now confidently reassign the ambiguous spacer peptides to the ID, β9 of D1, or the short inter-domain linker. Thus, with our reassigned cross-link designations we obtain a more accurate model of the Pf12-Pf41 interaction: Pf12 is in close proximity to the Pf41 ID as evidenced by ten crosslinks to this region, while cross-links between Pf12 and Pf41 D1 (three), D2 (five), or the C-terminal tail (three) were much fewer (Fig 5A), which is consistent with the ITC and protease protection data (Figs 3 and 4). While it is possible that the Pf41 ID solely contributes to the binding interface between Pf41 and Pf12, the cross-linking data combined with polymorphism analyses of Pf12 and Pf41 homologs are consistent with at least weak interactions between the 6-Cys domains of Pf41 and Pf12 to form an antiparallel interface [40, 41]. Moreover, our trypsin protease protection assay indicates that the Pf41 inter-domain linker (RSNNNVI) is stabilized in the presence of Pf12, indicating a tightening of the Pf41 6-Cys domains upon complex formation with Pf12 (Fig 4). Based on these data, we propose a refined model of the Pf12-Pf41 heterodimer, where the Pf41 ID serves as the key structural bridge to anchor the complex (Fig 5B).


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)

A refined Pf12-Pf41 heterodimeric assembly model.(A) Cross-linking designations revised from [15]; Pf41 ID specific interactions are bolded and shaded light grey. (B) Refined model of Pf12-Pf41 complex generated by manual docking and guided by cross-linking data. Pf12 is shown as a magenta surface, and Pf41 as a green surface except a model of the ID generated in iTASSER [42] displayed as a grey semi-transparent surface. The Pf41 ID model represents the relative size of this region compared to D1 and D2 and approximates how the ID could contact both Pf12 D1 and D2, but further studies probing the detailed structure and flexibility of this region are still needed. A previously identified Pf41 ID phosphorylation site [43] is shown in yellow and indicated by a yellow arrow. The black dotted line indicates uncertainty in the exact interface between the tandem 6-Cys domains of Pf12 and Pf41.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587554&req=5

pone.0139407.g005: A refined Pf12-Pf41 heterodimeric assembly model.(A) Cross-linking designations revised from [15]; Pf41 ID specific interactions are bolded and shaded light grey. (B) Refined model of Pf12-Pf41 complex generated by manual docking and guided by cross-linking data. Pf12 is shown as a magenta surface, and Pf41 as a green surface except a model of the ID generated in iTASSER [42] displayed as a grey semi-transparent surface. The Pf41 ID model represents the relative size of this region compared to D1 and D2 and approximates how the ID could contact both Pf12 D1 and D2, but further studies probing the detailed structure and flexibility of this region are still needed. A previously identified Pf41 ID phosphorylation site [43] is shown in yellow and indicated by a yellow arrow. The black dotted line indicates uncertainty in the exact interface between the tandem 6-Cys domains of Pf12 and Pf41.
Mentions: The structural and solution binding data reported here provided an opportunity to re-evaluate our previous Pf12-Pf41 cross-linking data [15]. Originally, cross-linked peptides were assigned to Pf41 D1, Linker, or D2 based on the previously predicted architecture of Pf41 with the spacer/linker region separating the two 6-Cys domains (Fig 1A). However, with the unambiguous domain assignments enabled by our Pf41 structure (Fig 3A), we can now confidently reassign the ambiguous spacer peptides to the ID, β9 of D1, or the short inter-domain linker. Thus, with our reassigned cross-link designations we obtain a more accurate model of the Pf12-Pf41 interaction: Pf12 is in close proximity to the Pf41 ID as evidenced by ten crosslinks to this region, while cross-links between Pf12 and Pf41 D1 (three), D2 (five), or the C-terminal tail (three) were much fewer (Fig 5A), which is consistent with the ITC and protease protection data (Figs 3 and 4). While it is possible that the Pf41 ID solely contributes to the binding interface between Pf41 and Pf12, the cross-linking data combined with polymorphism analyses of Pf12 and Pf41 homologs are consistent with at least weak interactions between the 6-Cys domains of Pf41 and Pf12 to form an antiparallel interface [40, 41]. Moreover, our trypsin protease protection assay indicates that the Pf41 inter-domain linker (RSNNNVI) is stabilized in the presence of Pf12, indicating a tightening of the Pf41 6-Cys domains upon complex formation with Pf12 (Fig 4). Based on these data, we propose a refined model of the Pf12-Pf41 heterodimer, where the Pf41 ID serves as the key structural bridge to anchor the complex (Fig 5B).

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