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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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E29A mutation in HflX is associated with the opening of a cleft between domains ND1 and ND2, as inferred by the exposure of an otherwise buried cysteine. (A) A schematic of domains in HflX is shown. Two salt bridges E29-R257 and Q28-N207 (indicated by dashed line) that affect the activities of ND1 (blue) and ND2 (red) are shown; these two domains are connected by a helical domain (yellow) that likely binds the ribosome. (B) A surface representation of SsHflX showing Ala18 buried at the interface of ND1 and ND2; the equivalent residue Ser32 in EcHflX was mutated to cysteine to create HflX-S32C mutant. (C) The proteins indicated on the X-axis of the histogram were incubated with DTNB, and the number of exposed cysteine residues, calculated as described in the ‘Materials and Methods’ section, is shown on the Y-axis. (D) MD simulations were run for 5 ns to gauge domain movements in HflX. A trajectory of domain movements in SsHflX during NVT simulation was obtained, of which conformations at three different stages of the simulation are shown. Red colored ribbon represents the initial protein conformation, white ribbon represents an intermediate frame in the trajectory and blue cartoon represents the conformation in the last frame at the end of 5 ns simulation. The inset shows how the domain opening would expose Ala18 (the corresponding S32 in EcHflX was mutated to a cysteine), which was initially buried at the interface of ND1 and ND2. Ala18 is shown as sticks (carbon atoms are green and hydrogen atoms are white).
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gkt705-F5: E29A mutation in HflX is associated with the opening of a cleft between domains ND1 and ND2, as inferred by the exposure of an otherwise buried cysteine. (A) A schematic of domains in HflX is shown. Two salt bridges E29-R257 and Q28-N207 (indicated by dashed line) that affect the activities of ND1 (blue) and ND2 (red) are shown; these two domains are connected by a helical domain (yellow) that likely binds the ribosome. (B) A surface representation of SsHflX showing Ala18 buried at the interface of ND1 and ND2; the equivalent residue Ser32 in EcHflX was mutated to cysteine to create HflX-S32C mutant. (C) The proteins indicated on the X-axis of the histogram were incubated with DTNB, and the number of exposed cysteine residues, calculated as described in the ‘Materials and Methods’ section, is shown on the Y-axis. (D) MD simulations were run for 5 ns to gauge domain movements in HflX. A trajectory of domain movements in SsHflX during NVT simulation was obtained, of which conformations at three different stages of the simulation are shown. Red colored ribbon represents the initial protein conformation, white ribbon represents an intermediate frame in the trajectory and blue cartoon represents the conformation in the last frame at the end of 5 ns simulation. The inset shows how the domain opening would expose Ala18 (the corresponding S32 in EcHflX was mutated to a cysteine), which was initially buried at the interface of ND1 and ND2. Ala18 is shown as sticks (carbon atoms are green and hydrogen atoms are white).

Mentions: The 5,5 -Dithio-bis(2-nitrobenzoic acid (DTNB) reactions with HflX mutants were carried out as described by Vopel et al. (26) with minor modifications. In all, 15 μM protein was incubated with 1 mM of colorless DTNB, (Invitrogen) or Ellman’s reagent (27), in degassed buffer containing 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 5 mM MgCl2, at 25°C. Formation of yellow thio-nitrobenzoate anion (TNB2−) was detected by measuring the absorbance at 412 nm using a UV-Vis Spectrophotometer (Perkin Elmer). A cysteine residue, on reaction with DTNB, generates one molecule of TNB2−. The number of reacting cysteine residues was calculated from the absorption values at 412 nm using an absorption coefficient of ε412 = 14150 M−1cm−1 for TNB2−. Accordingly, the ordinate scale in Figure 5C was converted from absorption values to reactive cysteine equivalents.


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)

E29A mutation in HflX is associated with the opening of a cleft between domains ND1 and ND2, as inferred by the exposure of an otherwise buried cysteine. (A) A schematic of domains in HflX is shown. Two salt bridges E29-R257 and Q28-N207 (indicated by dashed line) that affect the activities of ND1 (blue) and ND2 (red) are shown; these two domains are connected by a helical domain (yellow) that likely binds the ribosome. (B) A surface representation of SsHflX showing Ala18 buried at the interface of ND1 and ND2; the equivalent residue Ser32 in EcHflX was mutated to cysteine to create HflX-S32C mutant. (C) The proteins indicated on the X-axis of the histogram were incubated with DTNB, and the number of exposed cysteine residues, calculated as described in the ‘Materials and Methods’ section, is shown on the Y-axis. (D) MD simulations were run for 5 ns to gauge domain movements in HflX. A trajectory of domain movements in SsHflX during NVT simulation was obtained, of which conformations at three different stages of the simulation are shown. Red colored ribbon represents the initial protein conformation, white ribbon represents an intermediate frame in the trajectory and blue cartoon represents the conformation in the last frame at the end of 5 ns simulation. The inset shows how the domain opening would expose Ala18 (the corresponding S32 in EcHflX was mutated to a cysteine), which was initially buried at the interface of ND1 and ND2. Ala18 is shown as sticks (carbon atoms are green and hydrogen atoms are white).
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Related In: Results  -  Collection

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gkt705-F5: E29A mutation in HflX is associated with the opening of a cleft between domains ND1 and ND2, as inferred by the exposure of an otherwise buried cysteine. (A) A schematic of domains in HflX is shown. Two salt bridges E29-R257 and Q28-N207 (indicated by dashed line) that affect the activities of ND1 (blue) and ND2 (red) are shown; these two domains are connected by a helical domain (yellow) that likely binds the ribosome. (B) A surface representation of SsHflX showing Ala18 buried at the interface of ND1 and ND2; the equivalent residue Ser32 in EcHflX was mutated to cysteine to create HflX-S32C mutant. (C) The proteins indicated on the X-axis of the histogram were incubated with DTNB, and the number of exposed cysteine residues, calculated as described in the ‘Materials and Methods’ section, is shown on the Y-axis. (D) MD simulations were run for 5 ns to gauge domain movements in HflX. A trajectory of domain movements in SsHflX during NVT simulation was obtained, of which conformations at three different stages of the simulation are shown. Red colored ribbon represents the initial protein conformation, white ribbon represents an intermediate frame in the trajectory and blue cartoon represents the conformation in the last frame at the end of 5 ns simulation. The inset shows how the domain opening would expose Ala18 (the corresponding S32 in EcHflX was mutated to a cysteine), which was initially buried at the interface of ND1 and ND2. Ala18 is shown as sticks (carbon atoms are green and hydrogen atoms are white).
Mentions: The 5,5 -Dithio-bis(2-nitrobenzoic acid (DTNB) reactions with HflX mutants were carried out as described by Vopel et al. (26) with minor modifications. In all, 15 μM protein was incubated with 1 mM of colorless DTNB, (Invitrogen) or Ellman’s reagent (27), in degassed buffer containing 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 5 mM MgCl2, at 25°C. Formation of yellow thio-nitrobenzoate anion (TNB2−) was detected by measuring the absorbance at 412 nm using a UV-Vis Spectrophotometer (Perkin Elmer). A cysteine residue, on reaction with DTNB, generates one molecule of TNB2−. The number of reacting cysteine residues was calculated from the absorption values at 412 nm using an absorption coefficient of ε412 = 14150 M−1cm−1 for TNB2−. Accordingly, the ordinate scale in Figure 5C was converted from absorption values to reactive cysteine equivalents.

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