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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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Two salt bridges Q28-N207 and E29-R257 regulate GTPase and ATPase activities of EcHflX. (A) Residues constituting the salt bridges R114-D251 (blue asterisks), E29-R257 (black asterisks) and N207-Q28 (red asterisks) are shown in the multiple sequence alignment of representative HflX homologues (the corresponding gi numbers may be found in Supplementary Material). Numbers on the top (correspond to EcHflX) indicate regions containing these residues. Of these, E29, R114, D251, R257 and N207 are conserved among the HflX homologues. Q28 (first red asterisk from left) is not strictly conserved but is often a residue capable of forming a hydrogen bond via–OH group. (B) Interactions R114-D251, Q28-N207 and E29-R257 between ND2 (brown-red) and the ND1 (purple) in the homology model of EcHflX are shown. Disrupting the salt bridge R114-D251 did not affect ATP/GTP hydrolysis activities by EcHflX (see Supplementary Figure S4). (C) GTP (blue) and ATP (red) hydrolysis activities by HflX-WT and mutants HflX-Q28A and HflX-E29A are shown. Activity of HflX-WT was normalized to 100%. (D and E) Ability of HflX mutants to bind fluorescently labeled nucleotides, mant-ADP (D) and mant-GDP (E) was assessed by comparing fluorescence emission (arbitrary units) by the mant group in the presence and absence of the proteins, similarly as in Figure 2C. The inset shows the spectra recorded for the various constructs and are demarcated by corresponding numbers. Buffer control indicates only the buffer was present, and protein control implies the free protein (without mant-nucleotides).
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gkt705-F4: Two salt bridges Q28-N207 and E29-R257 regulate GTPase and ATPase activities of EcHflX. (A) Residues constituting the salt bridges R114-D251 (blue asterisks), E29-R257 (black asterisks) and N207-Q28 (red asterisks) are shown in the multiple sequence alignment of representative HflX homologues (the corresponding gi numbers may be found in Supplementary Material). Numbers on the top (correspond to EcHflX) indicate regions containing these residues. Of these, E29, R114, D251, R257 and N207 are conserved among the HflX homologues. Q28 (first red asterisk from left) is not strictly conserved but is often a residue capable of forming a hydrogen bond via–OH group. (B) Interactions R114-D251, Q28-N207 and E29-R257 between ND2 (brown-red) and the ND1 (purple) in the homology model of EcHflX are shown. Disrupting the salt bridge R114-D251 did not affect ATP/GTP hydrolysis activities by EcHflX (see Supplementary Figure S4). (C) GTP (blue) and ATP (red) hydrolysis activities by HflX-WT and mutants HflX-Q28A and HflX-E29A are shown. Activity of HflX-WT was normalized to 100%. (D and E) Ability of HflX mutants to bind fluorescently labeled nucleotides, mant-ADP (D) and mant-GDP (E) was assessed by comparing fluorescence emission (arbitrary units) by the mant group in the presence and absence of the proteins, similarly as in Figure 2C. The inset shows the spectra recorded for the various constructs and are demarcated by corresponding numbers. Buffer control indicates only the buffer was present, and protein control implies the free protein (without mant-nucleotides).

Mentions: Three stabilizing interactions in SsHflX seems to fasten the two domains, ND1 and ND2 together: these are (i) the carboxyl group of D232 (of ND2) that interacts with H97 (from ND1); (ii) the amino group of N189 (from ND2) that interacts with the carboxyl group of E14 (of ND1) and (iii) the amino group of R238 (from ND2) that interacts with carboxyl group of E15 (of ND1) (Supplementary Figure S2). Although the first and third are electrostatic interactions or salt bridges, the second is a hydrogen bond. However, for ease of presentation, we refer to all of these as salt bridges. To map these interactions in EcHflX, we generated a homology model based on the structure of SsHflX. A superposition of the two resulted in an root mean square deviation (r.m.s.d.) of 0.172, for 1077 backbone atoms. The salt bridges aforementioned would correspond to R114-D251, E29-R257 and Q28-N207 in EcHflX (Figure 4B). To assess their importance for ND1 and ND2, we disrupted them one at a time, by mutating one of the residues constituting a salt bridge. The mutants HflX-R114A, HflX-E29A and HflX-Q28A in EcHflX would thus disrupt the three salt bridges R114-D251, E29-R257 and Q28-N207, respectively. The salt-bridge disrupting mutants were generated, and mutant proteins, purified to homogeneity, were assayed for their ability to hydrolyze ATP and GTP.Figure 4.


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)

Two salt bridges Q28-N207 and E29-R257 regulate GTPase and ATPase activities of EcHflX. (A) Residues constituting the salt bridges R114-D251 (blue asterisks), E29-R257 (black asterisks) and N207-Q28 (red asterisks) are shown in the multiple sequence alignment of representative HflX homologues (the corresponding gi numbers may be found in Supplementary Material). Numbers on the top (correspond to EcHflX) indicate regions containing these residues. Of these, E29, R114, D251, R257 and N207 are conserved among the HflX homologues. Q28 (first red asterisk from left) is not strictly conserved but is often a residue capable of forming a hydrogen bond via–OH group. (B) Interactions R114-D251, Q28-N207 and E29-R257 between ND2 (brown-red) and the ND1 (purple) in the homology model of EcHflX are shown. Disrupting the salt bridge R114-D251 did not affect ATP/GTP hydrolysis activities by EcHflX (see Supplementary Figure S4). (C) GTP (blue) and ATP (red) hydrolysis activities by HflX-WT and mutants HflX-Q28A and HflX-E29A are shown. Activity of HflX-WT was normalized to 100%. (D and E) Ability of HflX mutants to bind fluorescently labeled nucleotides, mant-ADP (D) and mant-GDP (E) was assessed by comparing fluorescence emission (arbitrary units) by the mant group in the presence and absence of the proteins, similarly as in Figure 2C. The inset shows the spectra recorded for the various constructs and are demarcated by corresponding numbers. Buffer control indicates only the buffer was present, and protein control implies the free protein (without mant-nucleotides).
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gkt705-F4: Two salt bridges Q28-N207 and E29-R257 regulate GTPase and ATPase activities of EcHflX. (A) Residues constituting the salt bridges R114-D251 (blue asterisks), E29-R257 (black asterisks) and N207-Q28 (red asterisks) are shown in the multiple sequence alignment of representative HflX homologues (the corresponding gi numbers may be found in Supplementary Material). Numbers on the top (correspond to EcHflX) indicate regions containing these residues. Of these, E29, R114, D251, R257 and N207 are conserved among the HflX homologues. Q28 (first red asterisk from left) is not strictly conserved but is often a residue capable of forming a hydrogen bond via–OH group. (B) Interactions R114-D251, Q28-N207 and E29-R257 between ND2 (brown-red) and the ND1 (purple) in the homology model of EcHflX are shown. Disrupting the salt bridge R114-D251 did not affect ATP/GTP hydrolysis activities by EcHflX (see Supplementary Figure S4). (C) GTP (blue) and ATP (red) hydrolysis activities by HflX-WT and mutants HflX-Q28A and HflX-E29A are shown. Activity of HflX-WT was normalized to 100%. (D and E) Ability of HflX mutants to bind fluorescently labeled nucleotides, mant-ADP (D) and mant-GDP (E) was assessed by comparing fluorescence emission (arbitrary units) by the mant group in the presence and absence of the proteins, similarly as in Figure 2C. The inset shows the spectra recorded for the various constructs and are demarcated by corresponding numbers. Buffer control indicates only the buffer was present, and protein control implies the free protein (without mant-nucleotides).
Mentions: Three stabilizing interactions in SsHflX seems to fasten the two domains, ND1 and ND2 together: these are (i) the carboxyl group of D232 (of ND2) that interacts with H97 (from ND1); (ii) the amino group of N189 (from ND2) that interacts with the carboxyl group of E14 (of ND1) and (iii) the amino group of R238 (from ND2) that interacts with carboxyl group of E15 (of ND1) (Supplementary Figure S2). Although the first and third are electrostatic interactions or salt bridges, the second is a hydrogen bond. However, for ease of presentation, we refer to all of these as salt bridges. To map these interactions in EcHflX, we generated a homology model based on the structure of SsHflX. A superposition of the two resulted in an root mean square deviation (r.m.s.d.) of 0.172, for 1077 backbone atoms. The salt bridges aforementioned would correspond to R114-D251, E29-R257 and Q28-N207 in EcHflX (Figure 4B). To assess their importance for ND1 and ND2, we disrupted them one at a time, by mutating one of the residues constituting a salt bridge. The mutants HflX-R114A, HflX-E29A and HflX-Q28A in EcHflX would thus disrupt the three salt bridges R114-D251, E29-R257 and Q28-N207, respectively. The salt-bridge disrupting mutants were generated, and mutant proteins, purified to homogeneity, were assayed for their ability to hydrolyze ATP and GTP.Figure 4.

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