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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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Limited trypsinolysis of PfGyrA-CTD.Protein digests were visualised as a function of time. Time elapsed in minutes after initiation of reaction is indicated along the top of the gel. Decreases in band intensities of the original proteins (black triangles) are associated with reciprocal increases in band intensities of the fragments (white triangles). Edman degradation has identified the following amino acid sequences from the denoted fragments. Ec1, 572-IKEED-576; Ec2 1-GSHME-2 [underlined Gly-Ser-His residues derived from pET28a(+)]; Pf1, 855-LKFND-859 (also 857-FNDLQ-861 and 863-GNEQE-867 at lower intensities); Pf2, MKDHL (derived from the N-terminal His-tag).
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pone.0142313.g003: Limited trypsinolysis of PfGyrA-CTD.Protein digests were visualised as a function of time. Time elapsed in minutes after initiation of reaction is indicated along the top of the gel. Decreases in band intensities of the original proteins (black triangles) are associated with reciprocal increases in band intensities of the fragments (white triangles). Edman degradation has identified the following amino acid sequences from the denoted fragments. Ec1, 572-IKEED-576; Ec2 1-GSHME-2 [underlined Gly-Ser-His residues derived from pET28a(+)]; Pf1, 855-LKFND-859 (also 857-FNDLQ-861 and 863-GNEQE-867 at lower intensities); Pf2, MKDHL (derived from the N-terminal His-tag).

Mentions: Limited proteolysis of E. coli GyrA has shown that the C-terminal peptide bond of Arg571, located C-terminal to the GyrA-box in the loop of blade 1, is prone to tryptic digestion [40]. To probe the global conformation of PfGyrA-CTD, the protein was subjected to limited trypsinolysis and aliquots taken at various time points were separated using SDS-PAGE. An overall similar digestion pattern was observed for both His-PfGyrA-CTD and EcGyrA-CTD(WT) (Fig 3). The undigested proteins at T = 0 appeared as a single band which was digested into a larger and a smaller fragment. Proteins from these bands were subjected to further analyses by N-terminal sequencing and MALDI-TOF. Both results confirm cleavage to have occurred at the C-terminal end of Arg854 in His-PfGyrA-CTD, and also at Lys856 and Lys862. All three cleavages sites are located within a Plasmodium specific insertion (Fig 2A). From the small decrease in the molecular weight of band Pf1 combined with the appearance of no band other than Pf2, it is likely that cleavage at the C-terminus, does not occur or is minimal. Such specific, limited cleavage sites suggest that His-PfGyrA-CTD assumes a well-folded globular structure. Secondly, conformational similarity to E. coli GyrA-CTD at more local level is implied by the similarity in digestion patterns, and this notion is supported by the sequence alignment which indicates the cleavage sites to occur between α1 and β3 of blade 1 for both His-PfGyrA-CTD and EcGyrA-CTD(WT).


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)

Limited trypsinolysis of PfGyrA-CTD.Protein digests were visualised as a function of time. Time elapsed in minutes after initiation of reaction is indicated along the top of the gel. Decreases in band intensities of the original proteins (black triangles) are associated with reciprocal increases in band intensities of the fragments (white triangles). Edman degradation has identified the following amino acid sequences from the denoted fragments. Ec1, 572-IKEED-576; Ec2 1-GSHME-2 [underlined Gly-Ser-His residues derived from pET28a(+)]; Pf1, 855-LKFND-859 (also 857-FNDLQ-861 and 863-GNEQE-867 at lower intensities); Pf2, MKDHL (derived from the N-terminal His-tag).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142313.g003: Limited trypsinolysis of PfGyrA-CTD.Protein digests were visualised as a function of time. Time elapsed in minutes after initiation of reaction is indicated along the top of the gel. Decreases in band intensities of the original proteins (black triangles) are associated with reciprocal increases in band intensities of the fragments (white triangles). Edman degradation has identified the following amino acid sequences from the denoted fragments. Ec1, 572-IKEED-576; Ec2 1-GSHME-2 [underlined Gly-Ser-His residues derived from pET28a(+)]; Pf1, 855-LKFND-859 (also 857-FNDLQ-861 and 863-GNEQE-867 at lower intensities); Pf2, MKDHL (derived from the N-terminal His-tag).
Mentions: Limited proteolysis of E. coli GyrA has shown that the C-terminal peptide bond of Arg571, located C-terminal to the GyrA-box in the loop of blade 1, is prone to tryptic digestion [40]. To probe the global conformation of PfGyrA-CTD, the protein was subjected to limited trypsinolysis and aliquots taken at various time points were separated using SDS-PAGE. An overall similar digestion pattern was observed for both His-PfGyrA-CTD and EcGyrA-CTD(WT) (Fig 3). The undigested proteins at T = 0 appeared as a single band which was digested into a larger and a smaller fragment. Proteins from these bands were subjected to further analyses by N-terminal sequencing and MALDI-TOF. Both results confirm cleavage to have occurred at the C-terminal end of Arg854 in His-PfGyrA-CTD, and also at Lys856 and Lys862. All three cleavages sites are located within a Plasmodium specific insertion (Fig 2A). From the small decrease in the molecular weight of band Pf1 combined with the appearance of no band other than Pf2, it is likely that cleavage at the C-terminus, does not occur or is minimal. Such specific, limited cleavage sites suggest that His-PfGyrA-CTD assumes a well-folded globular structure. Secondly, conformational similarity to E. coli GyrA-CTD at more local level is implied by the similarity in digestion patterns, and this notion is supported by the sequence alignment which indicates the cleavage sites to occur between α1 and β3 of blade 1 for both His-PfGyrA-CTD and EcGyrA-CTD(WT).

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