Global conformational transitions in Escherichia coli primary replicative helicase DnaB protein induced by ATP, ADP, and single-stranded DNA binding

Multiple conformational states of the helicase hexamer

Maria J. Jezewska, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is S20,w = 11.9 ± 0.2 compared to the sedimentation coefficient S20,w = 10.5 ± 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 ± 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-d∈eA(p∈A)19, S20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a "closed" conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a "tense" state which binds ssDNA with an affinity of ∼4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a "relaxed" conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant ~3 orders of magnitude weaker than in the presence of the ATP analog (tense state).

Original languageEnglish (US)
Pages (from-to)4261-4265
Number of pages5
JournalJournal of Biological Chemistry
Volume271
Issue number8
StatePublished - Feb 23 1996

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DnaB Helicases
Single-Stranded DNA
Sedimentation
Adenosine Diphosphate
Escherichia coli
Adenosine Triphosphate
Adenylyl Imidodiphosphate
Conformations
Proteins
Nucleotides
Enzymes
Fluorescence
Titration

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Global conformational transitions in Escherichia coli primary replicative helicase DnaB protein induced by ATP, ADP, and single-stranded DNA binding: Multiple conformational states of the helicase hexamer",
abstract = "The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is S20,w = 11.9 ± 0.2 compared to the sedimentation coefficient S20,w = 10.5 ± 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 ± 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-d∈eA(p∈A)19, S20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a {"}closed{"} conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a {"}tense{"} state which binds ssDNA with an affinity of ∼4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a {"}relaxed{"} conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant ~3 orders of magnitude weaker than in the presence of the ATP analog (tense state).",
author = "Jezewska, {Maria J.} and Wlodzimierz Bujalowski",
year = "1996",
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T1 - Global conformational transitions in Escherichia coli primary replicative helicase DnaB protein induced by ATP, ADP, and single-stranded DNA binding

T2 - Multiple conformational states of the helicase hexamer

AU - Jezewska, Maria J.

AU - Bujalowski, Wlodzimierz

PY - 1996/2/23

Y1 - 1996/2/23

N2 - The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is S20,w = 11.9 ± 0.2 compared to the sedimentation coefficient S20,w = 10.5 ± 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 ± 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-d∈eA(p∈A)19, S20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a "closed" conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a "tense" state which binds ssDNA with an affinity of ∼4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a "relaxed" conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant ~3 orders of magnitude weaker than in the presence of the ATP analog (tense state).

AB - The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is S20,w = 11.9 ± 0.2 compared to the sedimentation coefficient S20,w = 10.5 ± 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 ± 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-d∈eA(p∈A)19, S20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a "closed" conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a "tense" state which binds ssDNA with an affinity of ∼4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a "relaxed" conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant ~3 orders of magnitude weaker than in the presence of the ATP analog (tense state).

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