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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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Schematic representation of the influence of the neighboring domains on the catalytic activities. (A) Effect of neighboring domains on the GTPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (negative regualtion depicted by ‘-’ sign) the GTPase activity of ND2. This is deduced based on the higher catalytic efficiency, Vmax/Km of HflX-ND2 (i.e. ND2 alone) as compared with that of HflX-ΔC (in which NTD and ND2 are present); compare efficiencies of these in the inset. A higher catalytic efficiency of HflX-ΔN as compared with HflX-FL also emphasizes the inhibitory role played by NTD. Compare efficiencies of these in the inset and see Table 1. (B) Influence of neighboring domains on the ATPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (depicted by ‘-’ sign) ATP hydroysis by ND2 as inferred from a higher activity by HflX-ND2 (i.e. ND2 alone) than by HflX-ΔC (which has NTD and ND2). Compare Vmax/Km of HflX-ND2 versus HflX-ΔC in the inset (also shown in Table 1). Similarly, CTD also negatively regulates ATP hydroysis of ND2 as inferred from a higher efficiency of HflX-ND2 than that of HflX-ΔN (which has ND2 and CTD). Compare these values in the inset. Furthermore, NTD and CTD appear to oppose each other’s inhibition and lead to an increased efficiency in HflX-FL, as compared with HflXΔC and HflX-ΔN, but not HflX-ND2 (compare Vmax/Km values of HflX-FL versus HflX-ΔC and HflX-ΔN in the inset and Table 1). However, ND2 alone (HflX-ND2) possesses the highest activity. Also note the relative difference in efficiencies for GTP and ATP hydrolysis, indicating HflX is a better ATPase.
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gkt705-F3: Schematic representation of the influence of the neighboring domains on the catalytic activities. (A) Effect of neighboring domains on the GTPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (negative regualtion depicted by ‘-’ sign) the GTPase activity of ND2. This is deduced based on the higher catalytic efficiency, Vmax/Km of HflX-ND2 (i.e. ND2 alone) as compared with that of HflX-ΔC (in which NTD and ND2 are present); compare efficiencies of these in the inset. A higher catalytic efficiency of HflX-ΔN as compared with HflX-FL also emphasizes the inhibitory role played by NTD. Compare efficiencies of these in the inset and see Table 1. (B) Influence of neighboring domains on the ATPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (depicted by ‘-’ sign) ATP hydroysis by ND2 as inferred from a higher activity by HflX-ND2 (i.e. ND2 alone) than by HflX-ΔC (which has NTD and ND2). Compare Vmax/Km of HflX-ND2 versus HflX-ΔC in the inset (also shown in Table 1). Similarly, CTD also negatively regulates ATP hydroysis of ND2 as inferred from a higher efficiency of HflX-ND2 than that of HflX-ΔN (which has ND2 and CTD). Compare these values in the inset. Furthermore, NTD and CTD appear to oppose each other’s inhibition and lead to an increased efficiency in HflX-FL, as compared with HflXΔC and HflX-ΔN, but not HflX-ND2 (compare Vmax/Km values of HflX-FL versus HflX-ΔC and HflX-ΔN in the inset and Table 1). However, ND2 alone (HflX-ND2) possesses the highest activity. Also note the relative difference in efficiencies for GTP and ATP hydrolysis, indicating HflX is a better ATPase.

Mentions: We further sought to probe how the neighboring domains regulate the hydrolysis activity of ND1. For this, we systematically determined the kinetic constants (for ATP, GTP hydrolysis separately) for constructs lacking one domain at a time (see Table 1). Indeed, we could identify a regulation imposed by the neighboring domains. To evaluate the influence of NTD (henceforth, referred as ND1 + HD), a deletion construct of EcHflX devoid of ND1 + HD (residues 1–192), i.e. HflX-ΔN was generated. Kinetic constants Km and Vmax were similarly determined. The Km (0.557 ± 0.136 mM) and Vmax (0.74 ± 0.02 min−1) values for GTP hydrolysis by HflX-ΔN are comparable with those for HflX-ND2, i.e. ND2 domain in isolation (see Table 1). This also implies that like HflX-ND2, HflX-ΔN also exhibits ∼4-fold higher efficiency as compared with full-length in hydrolyzing GTP. Put together, these clearly imply that in the full-length protein, ND1 + HD render an inhibitory effect on the GTP hydrolysis of ND2 (Figure 3A). These results are in concordance with studies on SsHflX, where a similar increase in activity was observed on deleting ND1 + HD (23).Figure 3.


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)

Schematic representation of the influence of the neighboring domains on the catalytic activities. (A) Effect of neighboring domains on the GTPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (negative regualtion depicted by ‘-’ sign) the GTPase activity of ND2. This is deduced based on the higher catalytic efficiency, Vmax/Km of HflX-ND2 (i.e. ND2 alone) as compared with that of HflX-ΔC (in which NTD and ND2 are present); compare efficiencies of these in the inset. A higher catalytic efficiency of HflX-ΔN as compared with HflX-FL also emphasizes the inhibitory role played by NTD. Compare efficiencies of these in the inset and see Table 1. (B) Influence of neighboring domains on the ATPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (depicted by ‘-’ sign) ATP hydroysis by ND2 as inferred from a higher activity by HflX-ND2 (i.e. ND2 alone) than by HflX-ΔC (which has NTD and ND2). Compare Vmax/Km of HflX-ND2 versus HflX-ΔC in the inset (also shown in Table 1). Similarly, CTD also negatively regulates ATP hydroysis of ND2 as inferred from a higher efficiency of HflX-ND2 than that of HflX-ΔN (which has ND2 and CTD). Compare these values in the inset. Furthermore, NTD and CTD appear to oppose each other’s inhibition and lead to an increased efficiency in HflX-FL, as compared with HflXΔC and HflX-ΔN, but not HflX-ND2 (compare Vmax/Km values of HflX-FL versus HflX-ΔC and HflX-ΔN in the inset and Table 1). However, ND2 alone (HflX-ND2) possesses the highest activity. Also note the relative difference in efficiencies for GTP and ATP hydrolysis, indicating HflX is a better ATPase.
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Related In: Results  -  Collection

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gkt705-F3: Schematic representation of the influence of the neighboring domains on the catalytic activities. (A) Effect of neighboring domains on the GTPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (negative regualtion depicted by ‘-’ sign) the GTPase activity of ND2. This is deduced based on the higher catalytic efficiency, Vmax/Km of HflX-ND2 (i.e. ND2 alone) as compared with that of HflX-ΔC (in which NTD and ND2 are present); compare efficiencies of these in the inset. A higher catalytic efficiency of HflX-ΔN as compared with HflX-FL also emphasizes the inhibitory role played by NTD. Compare efficiencies of these in the inset and see Table 1. (B) Influence of neighboring domains on the ATPase activity in EcHflX. NTD (ND1 + HD) negatively regulates (depicted by ‘-’ sign) ATP hydroysis by ND2 as inferred from a higher activity by HflX-ND2 (i.e. ND2 alone) than by HflX-ΔC (which has NTD and ND2). Compare Vmax/Km of HflX-ND2 versus HflX-ΔC in the inset (also shown in Table 1). Similarly, CTD also negatively regulates ATP hydroysis of ND2 as inferred from a higher efficiency of HflX-ND2 than that of HflX-ΔN (which has ND2 and CTD). Compare these values in the inset. Furthermore, NTD and CTD appear to oppose each other’s inhibition and lead to an increased efficiency in HflX-FL, as compared with HflXΔC and HflX-ΔN, but not HflX-ND2 (compare Vmax/Km values of HflX-FL versus HflX-ΔC and HflX-ΔN in the inset and Table 1). However, ND2 alone (HflX-ND2) possesses the highest activity. Also note the relative difference in efficiencies for GTP and ATP hydrolysis, indicating HflX is a better ATPase.
Mentions: We further sought to probe how the neighboring domains regulate the hydrolysis activity of ND1. For this, we systematically determined the kinetic constants (for ATP, GTP hydrolysis separately) for constructs lacking one domain at a time (see Table 1). Indeed, we could identify a regulation imposed by the neighboring domains. To evaluate the influence of NTD (henceforth, referred as ND1 + HD), a deletion construct of EcHflX devoid of ND1 + HD (residues 1–192), i.e. HflX-ΔN was generated. Kinetic constants Km and Vmax were similarly determined. The Km (0.557 ± 0.136 mM) and Vmax (0.74 ± 0.02 min−1) values for GTP hydrolysis by HflX-ΔN are comparable with those for HflX-ND2, i.e. ND2 domain in isolation (see Table 1). This also implies that like HflX-ND2, HflX-ΔN also exhibits ∼4-fold higher efficiency as compared with full-length in hydrolyzing GTP. Put together, these clearly imply that in the full-length protein, ND1 + HD render an inhibitory effect on the GTP hydrolysis of ND2 (Figure 3A). These results are in concordance with studies on SsHflX, where a similar increase in activity was observed on deleting ND1 + HD (23).Figure 3.

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