Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein

R. Galletto, S. Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct-binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the γ-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-γ-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are ~2 and ~4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADp are ~2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.

Original languageEnglish (US)
Pages (from-to)12959-12969
Number of pages11
JournalBiochemistry
Volume39
Issue number42
DOIs
StatePublished - Oct 24 2000

Fingerprint

Escherichia coli
Nucleotides
Adenosine Triphosphate
Proteins
Ribose
Stoichiometry
Adenosine Diphosphate
Binding Sites
Titration
Phosphates
Adenylyl Imidodiphosphate
Magnesium
Cations
Monomers
Fluorescence
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein. / Galletto, R.; Rajendran, S.; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 39, No. 42, 24.10.2000, p. 12959-12969.

Research output: Contribution to journalArticle

@article{0e329077b6e64c68a5638f85ae8286b5,
title = "Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein",
abstract = "Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct-binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the γ-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-γ-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are ~2 and ~4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADp are ~2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.",
author = "R. Galletto and S. Rajendran and Wlodzimierz Bujalowski",
year = "2000",
month = "10",
day = "24",
doi = "10.1021/bi0012484",
language = "English (US)",
volume = "39",
pages = "12959--12969",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "42",

}

TY - JOUR

T1 - Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein

AU - Galletto, R.

AU - Rajendran, S.

AU - Bujalowski, Wlodzimierz

PY - 2000/10/24

Y1 - 2000/10/24

N2 - Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct-binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the γ-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-γ-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are ~2 and ~4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADp are ~2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.

AB - Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct-binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the γ-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-γ-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are ~2 and ~4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADp are ~2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.

UR - http://www.scopus.com/inward/record.url?scp=0034711089&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034711089&partnerID=8YFLogxK

U2 - 10.1021/bi0012484

DO - 10.1021/bi0012484

M3 - Article

C2 - 11041861

AN - SCOPUS:0034711089

VL - 39

SP - 12959

EP - 12969

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 42

ER -