The nucleotide-binding site of the Escherichia coli DnaC protein

Molecular topography of DnaC protein-nucleotide cofactor complexes

Roberto Galletto, Maria J. Jezewska, Rodrigo Maillard, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3′-O-(N-methylantraniloyl)- 5′-triphosphate (MANT-ATP) and 3′-O-(N-methylantraniloyl)-5′- diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.

Original languageEnglish (US)
Pages (from-to)331-353
Number of pages23
JournalCell Biochemistry and Biophysics
Volume43
Issue number3
DOIs
StatePublished - Nov 2005

Fingerprint

Escherichia coli Proteins
Topography
Escherichia coli
Nucleotides
Binding Sites
Adenosine Triphosphate
Tryptophan
Proteins
Membrane Proteins
Adenosine Diphosphate
Tyrosine
Conformations
Diphosphates
Fluorescence Spectrometry
Fluorescence spectroscopy
Magnesium
Quenching
Fluorescence
Molecules

Keywords

  • DNA replication
  • DnaB helicase
  • DnaC protein
  • Motor proteins
  • Nucleotide binding

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Biophysics

Cite this

The nucleotide-binding site of the Escherichia coli DnaC protein : Molecular topography of DnaC protein-nucleotide cofactor complexes. / Galletto, Roberto; Jezewska, Maria J.; Maillard, Rodrigo; Bujalowski, Wlodzimierz.

In: Cell Biochemistry and Biophysics, Vol. 43, No. 3, 11.2005, p. 331-353.

Research output: Contribution to journalArticle

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