Limits...
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.

Show MeSH

Related in: MedlinePlus

a, domain representation of PfUIS3. b, soluble domain of PfUIS3 is dimeric in solution. Superdex 75 gel filtration analysis of PfUIS3 reveals the existence of dimers (blue) and monomers (red). c, SDS-PAGE of the fractions D, T, and M indicating truncation of the PfUIS3 construct from residues 89-229 (dimeric) to residues 130-229 (monomeric in nature). d, SDS-PAGE analysis of glutaraldehyde cross-linking (0.1%) control PfUIS3 (lane 1) and glutaraldehyde cross-linked PfUIS3 (lane 2). PfUIS3 at a concentration of 4 mg ml-1 was used for gel-filtration and cross-linking experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2527117&req=5

fig1: a, domain representation of PfUIS3. b, soluble domain of PfUIS3 is dimeric in solution. Superdex 75 gel filtration analysis of PfUIS3 reveals the existence of dimers (blue) and monomers (red). c, SDS-PAGE of the fractions D, T, and M indicating truncation of the PfUIS3 construct from residues 89-229 (dimeric) to residues 130-229 (monomeric in nature). d, SDS-PAGE analysis of glutaraldehyde cross-linking (0.1%) control PfUIS3 (lane 1) and glutaraldehyde cross-linked PfUIS3 (lane 2). PfUIS3 at a concentration of 4 mg ml-1 was used for gel-filtration and cross-linking experiments.

Mentions: PfUIS3 Production and Oligomeric State—UIS3 gene encoding for residues spanning the PfUIS3 soluble domain (83-229) (without the signal sequence, cytoplasmic tail, and the membrane anchor) was expressed in a bacterial overexpression system. Purified PfUIS3 (residues 83-229) was dimeric in solution as shown by protein cross-linking experiments and gel filtration chromatography (Fig. 1). Dimeric PfUIS3 (residues 83-229) failed to crystallize despite extensive screening; however, a monomeric form (residues 130-229) of PfUIS3 crystallized rapidly. Analysis of the PfUIS3 dimer-monomer transition using gel filtration chromatography and SDS-PAGE suggested that dimeric PfUIS3 underwent proteolysis and reduced to a stable, soluble domain spanning residues 130-229 (Fig. 1a). This suggests that residues within residues 83-129 of PfUIS3 are likely to be involved in dimerization of PfUIS3. From here on, this soluble domain will be referred to as PfUIS3130-229.


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)

a, domain representation of PfUIS3. b, soluble domain of PfUIS3 is dimeric in solution. Superdex 75 gel filtration analysis of PfUIS3 reveals the existence of dimers (blue) and monomers (red). c, SDS-PAGE of the fractions D, T, and M indicating truncation of the PfUIS3 construct from residues 89-229 (dimeric) to residues 130-229 (monomeric in nature). d, SDS-PAGE analysis of glutaraldehyde cross-linking (0.1%) control PfUIS3 (lane 1) and glutaraldehyde cross-linked PfUIS3 (lane 2). PfUIS3 at a concentration of 4 mg ml-1 was used for gel-filtration and cross-linking experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527117&req=5

fig1: a, domain representation of PfUIS3. b, soluble domain of PfUIS3 is dimeric in solution. Superdex 75 gel filtration analysis of PfUIS3 reveals the existence of dimers (blue) and monomers (red). c, SDS-PAGE of the fractions D, T, and M indicating truncation of the PfUIS3 construct from residues 89-229 (dimeric) to residues 130-229 (monomeric in nature). d, SDS-PAGE analysis of glutaraldehyde cross-linking (0.1%) control PfUIS3 (lane 1) and glutaraldehyde cross-linked PfUIS3 (lane 2). PfUIS3 at a concentration of 4 mg ml-1 was used for gel-filtration and cross-linking experiments.
Mentions: PfUIS3 Production and Oligomeric State—UIS3 gene encoding for residues spanning the PfUIS3 soluble domain (83-229) (without the signal sequence, cytoplasmic tail, and the membrane anchor) was expressed in a bacterial overexpression system. Purified PfUIS3 (residues 83-229) was dimeric in solution as shown by protein cross-linking experiments and gel filtration chromatography (Fig. 1). Dimeric PfUIS3 (residues 83-229) failed to crystallize despite extensive screening; however, a monomeric form (residues 130-229) of PfUIS3 crystallized rapidly. Analysis of the PfUIS3 dimer-monomer transition using gel filtration chromatography and SDS-PAGE suggested that dimeric PfUIS3 underwent proteolysis and reduced to a stable, soluble domain spanning residues 130-229 (Fig. 1a). This suggests that residues within residues 83-129 of PfUIS3 are likely to be involved in dimerization of PfUIS3. From here on, this soluble domain will be referred to as PfUIS3130-229.

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