Probing the Impact of the echinT C-Terminal Domain on Structure and Catalysis

Sanaa Bardaweel, James Pace, Tsui Fen Chou, Vivian Cody, Carston R Wagner

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P21 with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the Tm value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step, followed by a rate-limiting hydrolysis step, was conserved. Nevertheless, the ability of the C-terminal deletion mutants to hydrolyze lysyl-AMP generated by LysU was greatly impaired. Taken together, our results highlight the emerging role of the C-terminus in governing the catalytic function of Hints.

Original languageEnglish (US)
Pages (from-to)627-638
Number of pages12
JournalJournal of Molecular Biology
Volume404
Issue number4
DOIs
StatePublished - Dec 10 2010

Fingerprint

Catalysis
Escherichia coli
Bacteria
Gram-Positive Bacteria
Adenosine Monophosphate
Eukaryota
Gram-Negative Bacteria
Histidine
Carrier Proteins
Hydrolysis
Nucleotides
Observation
Genome
Electrons
Genes
Proteins

Keywords

  • C-terminal loop
  • Crystal structure
  • E. coli Hint
  • Histidine triad nucleotide binding protein (Hint)
  • Lysyl-tRNA synthetase (LysRS)

Cite this

Probing the Impact of the echinT C-Terminal Domain on Structure and Catalysis. / Bardaweel, Sanaa; Pace, James; Chou, Tsui Fen; Cody, Vivian; Wagner, Carston R.

In: Journal of Molecular Biology, Vol. 404, No. 4, 10.12.2010, p. 627-638.

Research output: Contribution to journalArticle

Bardaweel, Sanaa ; Pace, James ; Chou, Tsui Fen ; Cody, Vivian ; Wagner, Carston R. / Probing the Impact of the echinT C-Terminal Domain on Structure and Catalysis. In: Journal of Molecular Biology. 2010 ; Vol. 404, No. 4. pp. 627-638.
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AB - Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P21 with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the Tm value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step, followed by a rate-limiting hydrolysis step, was conserved. Nevertheless, the ability of the C-terminal deletion mutants to hydrolyze lysyl-AMP generated by LysU was greatly impaired. Taken together, our results highlight the emerging role of the C-terminus in governing the catalytic function of Hints.

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