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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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Related in: MedlinePlus

Electron density map and crystallographic structure of NBD94566–663 of P. yoelii.(A), The NBD94566–663 molecule with its electron density map (2Fo-Fc at 1.5σ level) (blue). (B), Crystallographic structure of the A (green) B (blue) and C (magenta) chains of NBD94566–663. (C)
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pone-0009146-g006: Electron density map and crystallographic structure of NBD94566–663 of P. yoelii.(A), The NBD94566–663 molecule with its electron density map (2Fo-Fc at 1.5σ level) (blue). (B), Crystallographic structure of the A (green) B (blue) and C (magenta) chains of NBD94566–663. (C)

Mentions: Both MAD and SAD techniques for structure solution were tried. Various programs like, SHELX [40], SOLVE [41], CNS [42] and CCP4 [43] were used to identify the heavy atom sites (Se). Reasonable statistics and good initial map could be achieved with the SAD technique and using the help of SHELX program [40]. The resolution cut-off used for Se site identification is 4.4 Å and the correlation co-efficient stood at 39.51% with the Pseudo-free CC of 71.47%. Peak wavelength (0.97898 Å) data was used for the SAD technique in which 12 out of 18 Se sites (6 SeMet in each molecule) could be identified by SHELXD [44] that were further refined by SHELXE [45]. These 12 Se-sites were used to phase the structure factor with SHELXE and the resulting electron densities were improved by solvent flattening with SOLOMON and density modification by DM from the CCP4 package and RESOLVE [46]. The helical region could be readily identified from the initial map and the model of the NBD94566–663 was built manually using the program COOT [47]. Several cycles of manual building and fitting were carried out by COOT in combination with restrained refinement using REFMAC5 [48] of the CCP4 suite, keeping the temperature factor at overall. Each time a simulated annealing omit map was calculated and was used for model building (Supplementary Figure S3). The electron density map for the main chain atoms is excellent showing the typical sausage like features for the helical regions (Fig. 6A), whereas for the side chain atoms no visible densities could be identified. Se positions from the anomalous map were used to trace the chains. The final refined model has an R-factor of 33.9% and a R-free of 37.32% with good stereochemistry as can be inferred from the Ramachandran plot statistics given by PROCHECK [49]. The detailed summary of the refinement statistics is given in Table 1. The final electron density map for the structure shows good density for most of the backbone residues except for the residues 1–6 and 73–75 in chain A (Fig. 6A), 1–12 and 73–75 in chain B and 1–13, 31–33 and 73–75 in chain C, respectively (Fig. 6B).


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)

Electron density map and crystallographic structure of NBD94566–663 of P. yoelii.(A), The NBD94566–663 molecule with its electron density map (2Fo-Fc at 1.5σ level) (blue). (B), Crystallographic structure of the A (green) B (blue) and C (magenta) chains of NBD94566–663. (C)
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009146-g006: Electron density map and crystallographic structure of NBD94566–663 of P. yoelii.(A), The NBD94566–663 molecule with its electron density map (2Fo-Fc at 1.5σ level) (blue). (B), Crystallographic structure of the A (green) B (blue) and C (magenta) chains of NBD94566–663. (C)
Mentions: Both MAD and SAD techniques for structure solution were tried. Various programs like, SHELX [40], SOLVE [41], CNS [42] and CCP4 [43] were used to identify the heavy atom sites (Se). Reasonable statistics and good initial map could be achieved with the SAD technique and using the help of SHELX program [40]. The resolution cut-off used for Se site identification is 4.4 Å and the correlation co-efficient stood at 39.51% with the Pseudo-free CC of 71.47%. Peak wavelength (0.97898 Å) data was used for the SAD technique in which 12 out of 18 Se sites (6 SeMet in each molecule) could be identified by SHELXD [44] that were further refined by SHELXE [45]. These 12 Se-sites were used to phase the structure factor with SHELXE and the resulting electron densities were improved by solvent flattening with SOLOMON and density modification by DM from the CCP4 package and RESOLVE [46]. The helical region could be readily identified from the initial map and the model of the NBD94566–663 was built manually using the program COOT [47]. Several cycles of manual building and fitting were carried out by COOT in combination with restrained refinement using REFMAC5 [48] of the CCP4 suite, keeping the temperature factor at overall. Each time a simulated annealing omit map was calculated and was used for model building (Supplementary Figure S3). The electron density map for the main chain atoms is excellent showing the typical sausage like features for the helical regions (Fig. 6A), whereas for the side chain atoms no visible densities could be identified. Se positions from the anomalous map were used to trace the chains. The final refined model has an R-factor of 33.9% and a R-free of 37.32% with good stereochemistry as can be inferred from the Ramachandran plot statistics given by PROCHECK [49]. The detailed summary of the refinement statistics is given in Table 1. The final electron density map for the structure shows good density for most of the backbone residues except for the residues 1–6 and 73–75 in chain A (Fig. 6A), 1–12 and 73–75 in chain B and 1–13, 31–33 and 73–75 in chain C, respectively (Fig. 6B).

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