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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

Binding traits of NBD94444–547 to fluorescently labeled MgATP ATTO-647N.(A) Normalized autocorrelation functions of MgATP ATTO-647N (B) obtained by increasing the quantity of NBD94444–547 (increased protein concentration from left to right). (C) Binding of NBD94444–547 to MgADP ATTO-647N. The nucleotide analogue is displayed as relative bound fraction versus protein concentration. The best fit to titration curve A and B are shown as a non-linear, logistic curve fit. (D) Influence of NBD-Cl to MgATP ATTO-647N binding traits of NBD94444–547. The best fits at titration curves of supplementary figure 1B are shown as a pharmacological dose-response curve with variable Hill slope.
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pone-0009146-g005: Binding traits of NBD94444–547 to fluorescently labeled MgATP ATTO-647N.(A) Normalized autocorrelation functions of MgATP ATTO-647N (B) obtained by increasing the quantity of NBD94444–547 (increased protein concentration from left to right). (C) Binding of NBD94444–547 to MgADP ATTO-647N. The nucleotide analogue is displayed as relative bound fraction versus protein concentration. The best fit to titration curve A and B are shown as a non-linear, logistic curve fit. (D) Influence of NBD-Cl to MgATP ATTO-647N binding traits of NBD94444–547. The best fits at titration curves of supplementary figure 1B are shown as a pharmacological dose-response curve with variable Hill slope.

Mentions: The proper structural folding and structure formation of NBD94444–547 enabled us to study the ability of this protein to bind nucleotides by fluorescence correlation spectroscopy using fluorescent ATP and ADP derivatives ATP ATTO-647N and ADP ATTO-647N, respectively. As a reference, the mean count rate per Cyanine 5 (Cy5) fluorophore was determined to be 32.5±0.4 kHz. Compared to Cy5, the value of ATP ATTO-647N was determined to be 27.9±0.8 kHz and 51.3±3.5 kHz for ADP ATTO-647N. Fitting the autocorrelation functions resulted in characteristic times of diffusion τD = 50±1.1 µs for Cy5, τD = 70.2±1.3 µs for ATP ATTO-647N and τD = 68.9±2.2 µs for ADP ATTO-647N. The autocorrelation curves of the fluorescent ATP analogue for ATP ATTO-647N and ADP ATTO-647N in the absence and presence of increased concentrations of NBD94444–547 are show in figure 5A and in supplementary figure S1A, respectively. The increase of the mean diffusion time τD was due to the increase in the mass of the diffusing particle, when fluorescently labelled nucleotide bound to NBD94444–547, which is apparent in the displayed autocorrelation curves with increased protein concentrations from left to right. A binding constant (Kd) of 228±2.3 µM for MgATP ATTO-647N and 331±1.8 µM for MgADP ATTO-647N bound to NBD94444–547 was determined (Fig. 5B, C). By contrast no nucleotide-binding could be observed in the constructs NBD94566–663 and NBD94674–793.


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)

Binding traits of NBD94444–547 to fluorescently labeled MgATP ATTO-647N.(A) Normalized autocorrelation functions of MgATP ATTO-647N (B) obtained by increasing the quantity of NBD94444–547 (increased protein concentration from left to right). (C) Binding of NBD94444–547 to MgADP ATTO-647N. The nucleotide analogue is displayed as relative bound fraction versus protein concentration. The best fit to titration curve A and B are shown as a non-linear, logistic curve fit. (D) Influence of NBD-Cl to MgATP ATTO-647N binding traits of NBD94444–547. The best fits at titration curves of supplementary figure 1B are shown as a pharmacological dose-response curve with variable Hill slope.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818847&req=5

pone-0009146-g005: Binding traits of NBD94444–547 to fluorescently labeled MgATP ATTO-647N.(A) Normalized autocorrelation functions of MgATP ATTO-647N (B) obtained by increasing the quantity of NBD94444–547 (increased protein concentration from left to right). (C) Binding of NBD94444–547 to MgADP ATTO-647N. The nucleotide analogue is displayed as relative bound fraction versus protein concentration. The best fit to titration curve A and B are shown as a non-linear, logistic curve fit. (D) Influence of NBD-Cl to MgATP ATTO-647N binding traits of NBD94444–547. The best fits at titration curves of supplementary figure 1B are shown as a pharmacological dose-response curve with variable Hill slope.
Mentions: The proper structural folding and structure formation of NBD94444–547 enabled us to study the ability of this protein to bind nucleotides by fluorescence correlation spectroscopy using fluorescent ATP and ADP derivatives ATP ATTO-647N and ADP ATTO-647N, respectively. As a reference, the mean count rate per Cyanine 5 (Cy5) fluorophore was determined to be 32.5±0.4 kHz. Compared to Cy5, the value of ATP ATTO-647N was determined to be 27.9±0.8 kHz and 51.3±3.5 kHz for ADP ATTO-647N. Fitting the autocorrelation functions resulted in characteristic times of diffusion τD = 50±1.1 µs for Cy5, τD = 70.2±1.3 µs for ATP ATTO-647N and τD = 68.9±2.2 µs for ADP ATTO-647N. The autocorrelation curves of the fluorescent ATP analogue for ATP ATTO-647N and ADP ATTO-647N in the absence and presence of increased concentrations of NBD94444–547 are show in figure 5A and in supplementary figure S1A, respectively. The increase of the mean diffusion time τD was due to the increase in the mass of the diffusing particle, when fluorescently labelled nucleotide bound to NBD94444–547, which is apparent in the displayed autocorrelation curves with increased protein concentrations from left to right. A binding constant (Kd) of 228±2.3 µM for MgATP ATTO-647N and 331±1.8 µM for MgADP ATTO-647N bound to NBD94444–547 was determined (Fig. 5B, C). By contrast no nucleotide-binding could be observed in the constructs NBD94566–663 and NBD94674–793.

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