Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method

Iraida E. Andreeva, Anasuya Roychowdhury, Michal R. Szymanski, Maria J. Jezewska, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg2+, the association of the cofactors proceeds as a sequential three-step process RepA hexamer + Nk1⇄kF-1(H-N)1k 2⇄k-2(H-N)2k3⇄k -3(H-N)3 The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)2, which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)2, while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

Original languageEnglish (US)
Pages (from-to)10620-10636
Number of pages17
JournalBiochemistry
Volume48
Issue number44
DOIs
StatePublished - Nov 10 2009

Fingerprint

Plasmids
Nucleotides
Fluorescence
Proteins
Adenosine Diphosphate
Adenosine Triphosphate
Phosphates
Binding Sites
Administrative data processing
Single-Stranded DNA
Adenosinetriphosphate
DNA
Equilibrium constants
Entropy
Magnesium
Cations
Enthalpy
Association reactions
Enzymes
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method. / Andreeva, Iraida E.; Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 48, No. 44, 10.11.2009, p. 10620-10636.

Research output: Contribution to journalArticle

Andreeva, Iraida E. ; Roychowdhury, Anasuya ; Szymanski, Michal R. ; Jezewska, Maria J. ; Bujalowski, Wlodzimierz. / Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method. In: Biochemistry. 2009 ; Vol. 48, No. 44. pp. 10620-10636.
@article{d6b8159e275a4f92ab13de33b3c1f169,
title = "Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method",
abstract = "The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg2+, the association of the cofactors proceeds as a sequential three-step process RepA hexamer + Nk1⇄kF-1(H-N)1k 2⇄k-2(H-N)2k3⇄k -3(H-N)3 The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)2, which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)2, while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.",
author = "Andreeva, {Iraida E.} and Anasuya Roychowdhury and Szymanski, {Michal R.} and Jezewska, {Maria J.} and Wlodzimierz Bujalowski",
year = "2009",
month = "11",
day = "10",
doi = "10.1021/bi900940q",
language = "English (US)",
volume = "48",
pages = "10620--10636",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "44",

}

TY - JOUR

T1 - Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method

AU - Andreeva, Iraida E.

AU - Roychowdhury, Anasuya

AU - Szymanski, Michal R.

AU - Jezewska, Maria J.

AU - Bujalowski, Wlodzimierz

PY - 2009/11/10

Y1 - 2009/11/10

N2 - The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg2+, the association of the cofactors proceeds as a sequential three-step process RepA hexamer + Nk1⇄kF-1(H-N)1k 2⇄k-2(H-N)2k3⇄k -3(H-N)3 The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)2, which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)2, while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

AB - The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg2+, the association of the cofactors proceeds as a sequential three-step process RepA hexamer + Nk1⇄kF-1(H-N)1k 2⇄k-2(H-N)2k3⇄k -3(H-N)3 The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)2, which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)2, while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

UR - http://www.scopus.com/inward/record.url?scp=70449436543&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70449436543&partnerID=8YFLogxK

U2 - 10.1021/bi900940q

DO - 10.1021/bi900940q

M3 - Article

VL - 48

SP - 10620

EP - 10636

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 44

ER -