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Crystal structure of soluble domain of malaria sporozoite protein UIS3 in complex with lipid.

Sharma A, Yogavel M, Akhouri RR, Gill J, Sharma A - J. Biol. Chem. (2008)

Bottom Line: We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein.The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids.Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi, India.

ABSTRACT
Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-A resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3(130-229)) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3(130-229) is a novel, compact, and all alpha-helical structure bound to one molecule of PE. The PfUIS3(130-229)-PE complex structure reveals a novel binding site with specific interactions between PfUIS3(130-229) and the PE head group. One acyl chain of PE wraps around part of PfUIS3(130-229) and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

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Lipid electron density in PfUIS3130-229-PE complex. a, distinctly Y-shaped electron density was observed in difference maps during the model refinement of PfUIS3130-229. The electron density maps shown are contoured at 2.0 σ (green) and 1.0 σ (blue) for Fo - Fc and 2Fo - Fc, respectively. A molecule of PE was fit into the additional Y-shaped density observed. The final 2Fo - Fc map is contoured at a 1.0 σ level. b, tandem MS analysis of bound PEs extracted from PfUIS3130-229.
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fig5: Lipid electron density in PfUIS3130-229-PE complex. a, distinctly Y-shaped electron density was observed in difference maps during the model refinement of PfUIS3130-229. The electron density maps shown are contoured at 2.0 σ (green) and 1.0 σ (blue) for Fo - Fc and 2Fo - Fc, respectively. A molecule of PE was fit into the additional Y-shaped density observed. The final 2Fo - Fc map is contoured at a 1.0 σ level. b, tandem MS analysis of bound PEs extracted from PfUIS3130-229.

Mentions: PE Forms a 1:1 Complex with PfUIS3130-229—During model refinement of PfUIS3130-229, difference electron density maps revealed regions of elongated Y-shaped electron density near residues Glu166, Gln168, Asn222, and Asn226 and between H1 and H4 helices close to the surface of PfUIS3130-229. The shape of the extra surface electron density (contoured at 2.0 and 1.0 σ levels for Fo - Fc and 2Fo - Fc, respectively; Fig. 5a) immediately suggested a potential lipid/fatty acid interaction. An in-depth analysis of this extra density, including docking of fatty acids and lipids, suggested that only lipids such as PE and phosphatidylcholine satisfactorily docked into the distinctly Y-shaped electron density (Fig. 5a). The presence of bound ligands was tested by tandem MS analysis of purified crystallization grade PfUIS3130-229. The phospholipid range in time-of-flight MS analysis was between 680 and 780 daltons. The shown tandem MS analysis of 716 daltons at collision energy 40 eV for 5 min shows expected fragments of PE. These data indicated the presence of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine at a molecular size of 716 daltons (Fig. 5b) bound to PfUIS3130-229. This is consistent with the fact that phosphatidylethanolamine is an abundant lipid of E. coli and constitutes ∼75% of membrane lipids (17). Given that PfUIS3 was overexpressed in E. coli, it is therefore likely that PE was naturally bound during UIS3 overproduction within the bacterial cell, as has been found in many previous studies of fatty acid-binding proteins. Confirmation of ligand identity by mass spectrometry allowed us to model PE molecules into the extra density on the surface of PfUIS3130-229. Structure of PE (Protein Data Bank entry 1lqv) shows a remarkable fit in the Y-shaped density and makes close contacts with side chains emanating from PfUIS3130-229 (Figs. 5a and 6). We were able to model PE phosphate head groups for each of the six monomers of PfUIS3130-229 in the asymmetric unit. The excellent quality of difference and 2Fo - Fc maps allowed us to model three complete and three partial chains of PE. The models were refined using crystallographic data to 2.5 Å resolution, and Rfree was monitored throughout the course of model building and refinement of the PfUIS3130-229-PE complex described here.


Crystal structure of soluble domain of malaria sporozoite protein UIS3 in complex with lipid.

Sharma A, Yogavel M, Akhouri RR, Gill J, Sharma A - J. Biol. Chem. (2008)

Lipid electron density in PfUIS3130-229-PE complex. a, distinctly Y-shaped electron density was observed in difference maps during the model refinement of PfUIS3130-229. The electron density maps shown are contoured at 2.0 σ (green) and 1.0 σ (blue) for Fo - Fc and 2Fo - Fc, respectively. A molecule of PE was fit into the additional Y-shaped density observed. The final 2Fo - Fc map is contoured at a 1.0 σ level. b, tandem MS analysis of bound PEs extracted from PfUIS3130-229.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527117&req=5

fig5: Lipid electron density in PfUIS3130-229-PE complex. a, distinctly Y-shaped electron density was observed in difference maps during the model refinement of PfUIS3130-229. The electron density maps shown are contoured at 2.0 σ (green) and 1.0 σ (blue) for Fo - Fc and 2Fo - Fc, respectively. A molecule of PE was fit into the additional Y-shaped density observed. The final 2Fo - Fc map is contoured at a 1.0 σ level. b, tandem MS analysis of bound PEs extracted from PfUIS3130-229.
Mentions: PE Forms a 1:1 Complex with PfUIS3130-229—During model refinement of PfUIS3130-229, difference electron density maps revealed regions of elongated Y-shaped electron density near residues Glu166, Gln168, Asn222, and Asn226 and between H1 and H4 helices close to the surface of PfUIS3130-229. The shape of the extra surface electron density (contoured at 2.0 and 1.0 σ levels for Fo - Fc and 2Fo - Fc, respectively; Fig. 5a) immediately suggested a potential lipid/fatty acid interaction. An in-depth analysis of this extra density, including docking of fatty acids and lipids, suggested that only lipids such as PE and phosphatidylcholine satisfactorily docked into the distinctly Y-shaped electron density (Fig. 5a). The presence of bound ligands was tested by tandem MS analysis of purified crystallization grade PfUIS3130-229. The phospholipid range in time-of-flight MS analysis was between 680 and 780 daltons. The shown tandem MS analysis of 716 daltons at collision energy 40 eV for 5 min shows expected fragments of PE. These data indicated the presence of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine at a molecular size of 716 daltons (Fig. 5b) bound to PfUIS3130-229. This is consistent with the fact that phosphatidylethanolamine is an abundant lipid of E. coli and constitutes ∼75% of membrane lipids (17). Given that PfUIS3 was overexpressed in E. coli, it is therefore likely that PE was naturally bound during UIS3 overproduction within the bacterial cell, as has been found in many previous studies of fatty acid-binding proteins. Confirmation of ligand identity by mass spectrometry allowed us to model PE molecules into the extra density on the surface of PfUIS3130-229. Structure of PE (Protein Data Bank entry 1lqv) shows a remarkable fit in the Y-shaped density and makes close contacts with side chains emanating from PfUIS3130-229 (Figs. 5a and 6). We were able to model PE phosphate head groups for each of the six monomers of PfUIS3130-229 in the asymmetric unit. The excellent quality of difference and 2Fo - Fc maps allowed us to model three complete and three partial chains of PE. The models were refined using crystallographic data to 2.5 Å resolution, and Rfree was monitored throughout the course of model building and refinement of the PfUIS3130-229-PE complex described here.

Bottom Line: We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein.The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids.Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi, India.

ABSTRACT
Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-A resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3(130-229)) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3(130-229) is a novel, compact, and all alpha-helical structure bound to one molecule of PE. The PfUIS3(130-229)-PE complex structure reveals a novel binding site with specific interactions between PfUIS3(130-229) and the PE head group. One acyl chain of PE wraps around part of PfUIS3(130-229) and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

Show MeSH
Related in: MedlinePlus