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Structural determination of functional units of the nucleotide binding domain (NBD94) of the reticulocyte binding protein Py235 of Plasmodium yoelii.

Grüber A, Manimekalai MS, Balakrishna AM, Hunke C, Jeyakanthan J, Preiser PR, Grüber G - PLoS ONE (2010)

Bottom Line: The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop.By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. ggrueber@ntu.edu.sg

ABSTRACT

Background: Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.

Methodology/principal findings: In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.

Conclusions: These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.

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Small-angle X-ray scattering data of NBD94444-547 of P. yoelii.(A) Experimental scattering data (o) and the fitting curves (—; green: experimental, red: calculated from ab initio model) for NBD94444–547 of P. yoelii. (B) The distance distribution function of the same protein. (C) Low resolution structure of NBD94444–547 in solution determined from SAXS data. The two shapes are rotated clockwise by around 90° along the Y-axis.
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pone-0009146-g003: Small-angle X-ray scattering data of NBD94444-547 of P. yoelii.(A) Experimental scattering data (o) and the fitting curves (—; green: experimental, red: calculated from ab initio model) for NBD94444–547 of P. yoelii. (B) The distance distribution function of the same protein. (C) Low resolution structure of NBD94444–547 in solution determined from SAXS data. The two shapes are rotated clockwise by around 90° along the Y-axis.

Mentions: The high purity allowed small-angle X-ray scattering (SAXS) experiments to be performed, with the aim to determine the first low resolution structures of the nucleotide-binding segment NBD94444–547 and NBD94674–793 in solution. SAXS patterns from solutions of both proteins were recorded as described in Materials and Methods to yield the final composite scattering curves in figure 3A and 4A, respectively, that both proteins are monodispersed in solution. The radius of gyration Rg of NBD94444–547 is 33.4±1 Å and the maximum dimension Dmax of the protein is 134±2 Å (Fig. 3B). The gross structure of NBD94444–547 was restored ab initio from the scattering pattern in figure 3A using the dummy residues modeling program GASBOR [37], which fitted well to the experimental data in the entire scattering range (a typical fit displayed in figure 3A, curve 2, has the discrepancy χ = 1.28). Ten independent reconstructions yielded reproducible models and the average model and the most probable model are displayed in figure 3C. NBD94444–547 appears as an elongated molecule with a length of 134 Å, composed of two more globular domains and a spiral-like segment of about 73.1 Å in length between both domains.


Structural determination of functional units of the nucleotide binding domain (NBD94) of the reticulocyte binding protein Py235 of Plasmodium yoelii.

Grüber A, Manimekalai MS, Balakrishna AM, Hunke C, Jeyakanthan J, Preiser PR, Grüber G - PLoS ONE (2010)

Small-angle X-ray scattering data of NBD94444-547 of P. yoelii.(A) Experimental scattering data (o) and the fitting curves (—; green: experimental, red: calculated from ab initio model) for NBD94444–547 of P. yoelii. (B) The distance distribution function of the same protein. (C) Low resolution structure of NBD94444–547 in solution determined from SAXS data. The two shapes are rotated clockwise by around 90° along the Y-axis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2818847&req=5

pone-0009146-g003: Small-angle X-ray scattering data of NBD94444-547 of P. yoelii.(A) Experimental scattering data (o) and the fitting curves (—; green: experimental, red: calculated from ab initio model) for NBD94444–547 of P. yoelii. (B) The distance distribution function of the same protein. (C) Low resolution structure of NBD94444–547 in solution determined from SAXS data. The two shapes are rotated clockwise by around 90° along the Y-axis.
Mentions: The high purity allowed small-angle X-ray scattering (SAXS) experiments to be performed, with the aim to determine the first low resolution structures of the nucleotide-binding segment NBD94444–547 and NBD94674–793 in solution. SAXS patterns from solutions of both proteins were recorded as described in Materials and Methods to yield the final composite scattering curves in figure 3A and 4A, respectively, that both proteins are monodispersed in solution. The radius of gyration Rg of NBD94444–547 is 33.4±1 Å and the maximum dimension Dmax of the protein is 134±2 Å (Fig. 3B). The gross structure of NBD94444–547 was restored ab initio from the scattering pattern in figure 3A using the dummy residues modeling program GASBOR [37], which fitted well to the experimental data in the entire scattering range (a typical fit displayed in figure 3A, curve 2, has the discrepancy χ = 1.28). Ten independent reconstructions yielded reproducible models and the average model and the most probable model are displayed in figure 3C. NBD94444–547 appears as an elongated molecule with a length of 134 Å, composed of two more globular domains and a spiral-like segment of about 73.1 Å in length between both domains.

Bottom Line: The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop.By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. ggrueber@ntu.edu.sg

ABSTRACT

Background: Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.

Methodology/principal findings: In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.

Conclusions: These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.

Show MeSH
Related in: MedlinePlus