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Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase.

Jain N, Vithani N, Rafay A, Prakash B - Nucleic Acids Res. (2013)

Bottom Line: It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements.Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2.Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208106, India.

ABSTRACT
A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other's hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.

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The N terminal domain, ND1 in HflX is an NTP binding domain. (A) Domain organization in EcHflX, which has an additional CTD. (B) ND1 of SsHflX (sea green) (2qth) is superimposed onto the ATP-binding domain of DgkB (pdb ID: 2qv7) (golden-yellow). ATP bound to DgkB is shown in sticks. (C) Fluorescent nucleotide binding experiments carried out for HflX (i.e. full length) and HflX-ND1 (1–120 residues) are shown. mant-ATP binding was monitored by measuring fluorescence emission [380–600 nm; 5µM protein and 1 µM mant-ATP was used]. The spectra are labeled and color-coded as indicated in the inset. (D) ATP hydrolysis by HflX-ND1 was measured using radiolabeled α[32P]-ATP as described in ‘Materials and Methods’ section. The reaction was carried out for 60 min, at varying concentrations of the substrate (S); for each concentration, rate of the reaction (V) was calculated and a Lineweaver–Burk plot (plot between 1/S and 1/V), shown on the right, was used to determine Vmax and Km. (E) ATP hydrolysis was carried out in the presence and absence of Mg2+ ions. EDTA was added to achieve an Mg2+ free state. Activity in presence of Mg2+ was normalized to 100%.
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gkt705-F2: The N terminal domain, ND1 in HflX is an NTP binding domain. (A) Domain organization in EcHflX, which has an additional CTD. (B) ND1 of SsHflX (sea green) (2qth) is superimposed onto the ATP-binding domain of DgkB (pdb ID: 2qv7) (golden-yellow). ATP bound to DgkB is shown in sticks. (C) Fluorescent nucleotide binding experiments carried out for HflX (i.e. full length) and HflX-ND1 (1–120 residues) are shown. mant-ATP binding was monitored by measuring fluorescence emission [380–600 nm; 5µM protein and 1 µM mant-ATP was used]. The spectra are labeled and color-coded as indicated in the inset. (D) ATP hydrolysis by HflX-ND1 was measured using radiolabeled α[32P]-ATP as described in ‘Materials and Methods’ section. The reaction was carried out for 60 min, at varying concentrations of the substrate (S); for each concentration, rate of the reaction (V) was calculated and a Lineweaver–Burk plot (plot between 1/S and 1/V), shown on the right, was used to determine Vmax and Km. (E) ATP hydrolysis was carried out in the presence and absence of Mg2+ ions. EDTA was added to achieve an Mg2+ free state. Activity in presence of Mg2+ was normalized to 100%.

Mentions: To understand how HflX could satisfy ATP hydrolysis, we undertook characterization of the NTD and CTD. However, as NTD, unlike CTD, is well conserved, we focused on understanding its role; in EcHflX, this domain comprises residues 1–192. We carefully analyzed the only available structure of HflX, i.e. SsHflX from S. solfataricus, which is an atypical member lacking the CTD. NTD in SsHflx is 179 residues long and amino acids 166 to 179 is disordered in the available crystal structure (Figure 1) Inspecting this region suggested that NTD in SsHflX could further be divided into two distinct domains. The first domain (residues 1–98 and named ND1; for NTP-binding domain 1, see later in the text) closely resembles the Rossman fold and is made up of a centrally placed parallel β-sheet surrounded by 4 α-helices (Figure 1). The second, which we call the helical domain (HD: residues 99–165), is made up of two long α-helices. A significant part of HD (residues 123–143) is disordered in the structure of SsHflX (Figure 1). With this, EcHflX contains four domains arranged in the following order from N to C terminus: ND1-HD-ND2-CTD (Figure 2A).Figure 1.


Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase.

Jain N, Vithani N, Rafay A, Prakash B - Nucleic Acids Res. (2013)

The N terminal domain, ND1 in HflX is an NTP binding domain. (A) Domain organization in EcHflX, which has an additional CTD. (B) ND1 of SsHflX (sea green) (2qth) is superimposed onto the ATP-binding domain of DgkB (pdb ID: 2qv7) (golden-yellow). ATP bound to DgkB is shown in sticks. (C) Fluorescent nucleotide binding experiments carried out for HflX (i.e. full length) and HflX-ND1 (1–120 residues) are shown. mant-ATP binding was monitored by measuring fluorescence emission [380–600 nm; 5µM protein and 1 µM mant-ATP was used]. The spectra are labeled and color-coded as indicated in the inset. (D) ATP hydrolysis by HflX-ND1 was measured using radiolabeled α[32P]-ATP as described in ‘Materials and Methods’ section. The reaction was carried out for 60 min, at varying concentrations of the substrate (S); for each concentration, rate of the reaction (V) was calculated and a Lineweaver–Burk plot (plot between 1/S and 1/V), shown on the right, was used to determine Vmax and Km. (E) ATP hydrolysis was carried out in the presence and absence of Mg2+ ions. EDTA was added to achieve an Mg2+ free state. Activity in presence of Mg2+ was normalized to 100%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814362&req=5

gkt705-F2: The N terminal domain, ND1 in HflX is an NTP binding domain. (A) Domain organization in EcHflX, which has an additional CTD. (B) ND1 of SsHflX (sea green) (2qth) is superimposed onto the ATP-binding domain of DgkB (pdb ID: 2qv7) (golden-yellow). ATP bound to DgkB is shown in sticks. (C) Fluorescent nucleotide binding experiments carried out for HflX (i.e. full length) and HflX-ND1 (1–120 residues) are shown. mant-ATP binding was monitored by measuring fluorescence emission [380–600 nm; 5µM protein and 1 µM mant-ATP was used]. The spectra are labeled and color-coded as indicated in the inset. (D) ATP hydrolysis by HflX-ND1 was measured using radiolabeled α[32P]-ATP as described in ‘Materials and Methods’ section. The reaction was carried out for 60 min, at varying concentrations of the substrate (S); for each concentration, rate of the reaction (V) was calculated and a Lineweaver–Burk plot (plot between 1/S and 1/V), shown on the right, was used to determine Vmax and Km. (E) ATP hydrolysis was carried out in the presence and absence of Mg2+ ions. EDTA was added to achieve an Mg2+ free state. Activity in presence of Mg2+ was normalized to 100%.
Mentions: To understand how HflX could satisfy ATP hydrolysis, we undertook characterization of the NTD and CTD. However, as NTD, unlike CTD, is well conserved, we focused on understanding its role; in EcHflX, this domain comprises residues 1–192. We carefully analyzed the only available structure of HflX, i.e. SsHflX from S. solfataricus, which is an atypical member lacking the CTD. NTD in SsHflx is 179 residues long and amino acids 166 to 179 is disordered in the available crystal structure (Figure 1) Inspecting this region suggested that NTD in SsHflX could further be divided into two distinct domains. The first domain (residues 1–98 and named ND1; for NTP-binding domain 1, see later in the text) closely resembles the Rossman fold and is made up of a centrally placed parallel β-sheet surrounded by 4 α-helices (Figure 1). The second, which we call the helical domain (HD: residues 99–165), is made up of two long α-helices. A significant part of HD (residues 123–143) is disordered in the structure of SsHflX (Figure 1). With this, EcHflX contains four domains arranged in the following order from N to C terminus: ND1-HD-ND2-CTD (Figure 2A).Figure 1.

Bottom Line: It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements.Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2.Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208106, India.

ABSTRACT
A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other's hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.

Show MeSH
Related in: MedlinePlus