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Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA.

Nagano S, Seki E, Lin TY, Shirouzu M, Yokoyama S, Heddle JG - PLoS ONE (2015)

Bottom Line: However, it has proved difficult to obtain soluble protein.Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form.Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.

View Article: PubMed Central - PubMed

Affiliation: Heddle Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan.

ABSTRACT
Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.

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DNA gyrase structure.A Schematic of DNA gyrase bound to DNA prior to ATP binding. DNA is shown in black. The N-terminus of GyrB is shown in red and the C-terminus in grey and yellow. The N and C-termini of GyrA are shown in green and blue respectively. B Schematic of domains in gyrase proteins and constructs used in the present study. β-pinwheel blades are numbered for E. coli GyrA. Colouring scheme is as follows; N-terminal domain, green; MBP, orange; β-pinwheel motifs identified by the Pfam server [5], light-blue; putative β-pinwheel motifs, dark-blue; signal/transit peptide, grey; acidic C-terminal tail, black. Segments with diagonal fill indicate the Asn-rich region (residues 887–902). Numbers refer to amino acid positions (see text).
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pone.0142313.g001: DNA gyrase structure.A Schematic of DNA gyrase bound to DNA prior to ATP binding. DNA is shown in black. The N-terminus of GyrB is shown in red and the C-terminus in grey and yellow. The N and C-termini of GyrA are shown in green and blue respectively. B Schematic of domains in gyrase proteins and constructs used in the present study. β-pinwheel blades are numbered for E. coli GyrA. Colouring scheme is as follows; N-terminal domain, green; MBP, orange; β-pinwheel motifs identified by the Pfam server [5], light-blue; putative β-pinwheel motifs, dark-blue; signal/transit peptide, grey; acidic C-terminal tail, black. Segments with diagonal fill indicate the Asn-rich region (residues 887–902). Numbers refer to amino acid positions (see text).

Mentions: DNA gyrase is a type II topoisomerase (type II topo) with the unique capability of introducing negative supercoils into DNA (Fig 1A). It consists of two proteins, GyrA and GyrB, which form an A2B2 complex in the functioning enzyme whose arrangement and overall structure is known at low resolution [1]. Gyrase is found in prokaryotes and some lower eukaryotes and it is often accompanied by topoisomerase IV (topo IV) a second type II topo that possesses complementary activity, showing a preference for decatenating topologically linked DNAs and relaxing supercoiled DNA [2]. Like gyrase, topo IV is a heterotetramer with ParC and ParE proteins being the equivalent of GyrA and GyrB respectively. Topo IV can relax both positively and negatively supercoiled DNA, but the former is the preferred substrate. Despite the complementary activities of gyrase and topo IV, some organisms possess gyrase as the sole type II topo (for example Mycobacterium tuberculosis [3, 4]).


Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA.

Nagano S, Seki E, Lin TY, Shirouzu M, Yokoyama S, Heddle JG - PLoS ONE (2015)

DNA gyrase structure.A Schematic of DNA gyrase bound to DNA prior to ATP binding. DNA is shown in black. The N-terminus of GyrB is shown in red and the C-terminus in grey and yellow. The N and C-termini of GyrA are shown in green and blue respectively. B Schematic of domains in gyrase proteins and constructs used in the present study. β-pinwheel blades are numbered for E. coli GyrA. Colouring scheme is as follows; N-terminal domain, green; MBP, orange; β-pinwheel motifs identified by the Pfam server [5], light-blue; putative β-pinwheel motifs, dark-blue; signal/transit peptide, grey; acidic C-terminal tail, black. Segments with diagonal fill indicate the Asn-rich region (residues 887–902). Numbers refer to amino acid positions (see text).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643928&req=5

pone.0142313.g001: DNA gyrase structure.A Schematic of DNA gyrase bound to DNA prior to ATP binding. DNA is shown in black. The N-terminus of GyrB is shown in red and the C-terminus in grey and yellow. The N and C-termini of GyrA are shown in green and blue respectively. B Schematic of domains in gyrase proteins and constructs used in the present study. β-pinwheel blades are numbered for E. coli GyrA. Colouring scheme is as follows; N-terminal domain, green; MBP, orange; β-pinwheel motifs identified by the Pfam server [5], light-blue; putative β-pinwheel motifs, dark-blue; signal/transit peptide, grey; acidic C-terminal tail, black. Segments with diagonal fill indicate the Asn-rich region (residues 887–902). Numbers refer to amino acid positions (see text).
Mentions: DNA gyrase is a type II topoisomerase (type II topo) with the unique capability of introducing negative supercoils into DNA (Fig 1A). It consists of two proteins, GyrA and GyrB, which form an A2B2 complex in the functioning enzyme whose arrangement and overall structure is known at low resolution [1]. Gyrase is found in prokaryotes and some lower eukaryotes and it is often accompanied by topoisomerase IV (topo IV) a second type II topo that possesses complementary activity, showing a preference for decatenating topologically linked DNAs and relaxing supercoiled DNA [2]. Like gyrase, topo IV is a heterotetramer with ParC and ParE proteins being the equivalent of GyrA and GyrB respectively. Topo IV can relax both positively and negatively supercoiled DNA, but the former is the preferred substrate. Despite the complementary activities of gyrase and topo IV, some organisms possess gyrase as the sole type II topo (for example Mycobacterium tuberculosis [3, 4]).

Bottom Line: However, it has proved difficult to obtain soluble protein.Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form.Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.

View Article: PubMed Central - PubMed

Affiliation: Heddle Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan.

ABSTRACT
Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.

Show MeSH
Related in: MedlinePlus