Functional study on GTP hydrolysis by the GTP-binding protein from Sulfolobus solfataricus, a member of the HflX family

B. Huang, H. Wu, N. Hao, F. Blombach, J. van der Oost, X. Li, X.C. Zhang, Z. Rao

Research output: Contribution to journalArticleAcademicpeer-review

9 Citations (Scopus)

Abstract

GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment
LanguageEnglish
Pages103-113
JournalJournal of Biochemistry
Volume148
Issue number1
DOIs
Publication statusPublished - 2010

Fingerprint

Sulfolobus solfataricus
GTP Phosphohydrolases
Guanosine Triphosphate
GTP-Binding Proteins
Hydrolysis
ras Proteins
Archaea
Glutamine
Phenylalanine
Mutagenesis
Point Mutation
Amides
Hot Temperature
Thermodynamic stability
Crystal structure
Switches
Water
Molecules
Proteins

Keywords

  • ras p21
  • activation
  • mutants
  • gtpases
  • loop
  • substitutions
  • purification
  • resolution
  • software
  • complex

Cite this

Huang, B. ; Wu, H. ; Hao, N. ; Blombach, F. ; van der Oost, J. ; Li, X. ; Zhang, X.C. ; Rao, Z. / Functional study on GTP hydrolysis by the GTP-binding protein from Sulfolobus solfataricus, a member of the HflX family. In: Journal of Biochemistry. 2010 ; Vol. 148, No. 1. pp. 103-113.
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abstract = "GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment",
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Functional study on GTP hydrolysis by the GTP-binding protein from Sulfolobus solfataricus, a member of the HflX family. / Huang, B.; Wu, H.; Hao, N.; Blombach, F.; van der Oost, J.; Li, X.; Zhang, X.C.; Rao, Z.

In: Journal of Biochemistry, Vol. 148, No. 1, 2010, p. 103-113.

Research output: Contribution to journalArticleAcademicpeer-review

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N2 - GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment

AB - GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment

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