Limits...
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.

Show MeSH

Related in: MedlinePlus

Circular dichroism (CD) spectroscopy of the recombinant proteins.(A–C), Far-UV CD spectra of NBD94444–547 (A), NBD94566–663 (B) as well as NBD94674–793 (C), respectively. The SDS-gel in the insets show a sample of the corresponding purified constructs NBD94444–547, NBD94566–663 and NBD94674–793.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2818847&req=5

pone-0009146-g002: Circular dichroism (CD) spectroscopy of the recombinant proteins.(A–C), Far-UV CD spectra of NBD94444–547 (A), NBD94566–663 (B) as well as NBD94674–793 (C), respectively. The SDS-gel in the insets show a sample of the corresponding purified constructs NBD94444–547, NBD94566–663 and NBD94674–793.

Mentions: In order to understand the events of nucleotide-binding in NBD94 and its concerted structural alteration(s) in the C-terminal hinge region, as well as to determine the smallest segment of NBD94, still able to bind nucleotide, and thereby forming an attractive target for therapeutic agents, the truncated forms NBD94444–547, NBD94566–663 and NBD94674–793 have been generated (Fig. 1), in which predicted α-helical structures have been taken into account (see below). The SDS-PAGE of the produced recombinant NBD94444–547, NBD94566–663 and NBD94674–793 of Plasmodium yoelii revealed a prominent band of 13.2 kDa, 12.8 kDa and 15.2 kDa, respectively, which was found entirely within the soluble fraction. A Ni2+-NTA resin column and an imidazole-gradient were used to separate NBD94444–547, NBD94566–663 and NBD94674–793, respectively, from the main contaminating proteins. The protein NBD94444–547, NBD94566–663 and NBD94674–793, respectively, eluting at 125–300 mM imidazole were collected, concentrated and subsequently applied to a size exclusion column (Superdex 75 HR 10/30 column). Analysis of the isolated protein by SDS-PAGE revealed the high purity of NBD94444–547, NBD94566–663 and NBD94674–793 (Fig. 2A–C).


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)

Circular dichroism (CD) spectroscopy of the recombinant proteins.(A–C), Far-UV CD spectra of NBD94444–547 (A), NBD94566–663 (B) as well as NBD94674–793 (C), respectively. The SDS-gel in the insets show a sample of the corresponding purified constructs NBD94444–547, NBD94566–663 and NBD94674–793.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009146-g002: Circular dichroism (CD) spectroscopy of the recombinant proteins.(A–C), Far-UV CD spectra of NBD94444–547 (A), NBD94566–663 (B) as well as NBD94674–793 (C), respectively. The SDS-gel in the insets show a sample of the corresponding purified constructs NBD94444–547, NBD94566–663 and NBD94674–793.
Mentions: In order to understand the events of nucleotide-binding in NBD94 and its concerted structural alteration(s) in the C-terminal hinge region, as well as to determine the smallest segment of NBD94, still able to bind nucleotide, and thereby forming an attractive target for therapeutic agents, the truncated forms NBD94444–547, NBD94566–663 and NBD94674–793 have been generated (Fig. 1), in which predicted α-helical structures have been taken into account (see below). The SDS-PAGE of the produced recombinant NBD94444–547, NBD94566–663 and NBD94674–793 of Plasmodium yoelii revealed a prominent band of 13.2 kDa, 12.8 kDa and 15.2 kDa, respectively, which was found entirely within the soluble fraction. A Ni2+-NTA resin column and an imidazole-gradient were used to separate NBD94444–547, NBD94566–663 and NBD94674–793, respectively, from the main contaminating proteins. The protein NBD94444–547, NBD94566–663 and NBD94674–793, respectively, eluting at 125–300 mM imidazole were collected, concentrated and subsequently applied to a size exclusion column (Superdex 75 HR 10/30 column). Analysis of the isolated protein by SDS-PAGE revealed the high purity of NBD94444–547, NBD94566–663 and NBD94674–793 (Fig. 2A–C).

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