ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase hexamer formation at low micromolar concentrations

Agnieszka M. Kuriata, Manas Chakraborty, J. Nathan Henderson, Suratna Hazra, Andrew J. Serban, Tuong V T Pham, Marcia Levitus, Rebekka Wachter

Research output: Contribution to journalArticle

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Abstract

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

Original languageEnglish (US)
Pages (from-to)7232-7246
Number of pages15
JournalBiochemistry
Volume53
Issue number46
DOIs
StatePublished - Nov 25 2014

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Oxygenases
Ribulose-Bisphosphate Carboxylase
Tissue Plasminogen Activator
Magnesium
Cotton
Adenosine Diphosphate
Adenosine Triphosphate
Fluorescence Spectrometry
Fluorescence
Spectroscopy
Carbon Cycle
Equilibrium constants
Titration
Thermodynamics
Dimers
Adenosine Triphosphatases
ribulose-1,5 diphosphate
Cations
Proteins
Carbon

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

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ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase hexamer formation at low micromolar concentrations. / Kuriata, Agnieszka M.; Chakraborty, Manas; Henderson, J. Nathan; Hazra, Suratna; Serban, Andrew J.; Pham, Tuong V T; Levitus, Marcia; Wachter, Rebekka.

In: Biochemistry, Vol. 53, No. 46, 25.11.2014, p. 7232-7246.

Research output: Contribution to journalArticle

Kuriata, Agnieszka M. ; Chakraborty, Manas ; Henderson, J. Nathan ; Hazra, Suratna ; Serban, Andrew J. ; Pham, Tuong V T ; Levitus, Marcia ; Wachter, Rebekka. / ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase hexamer formation at low micromolar concentrations. In: Biochemistry. 2014 ; Vol. 53, No. 46. pp. 7232-7246.
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abstract = "We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80{\%} of subunits with ATPγS, compared with just 30-40{\%} with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18{\%} with ATPγS and 26-28{\%} with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10{\%} of total protein with ATPγS and ∼25{\%} with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.",
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T1 - ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase hexamer formation at low micromolar concentrations

AU - Kuriata, Agnieszka M.

AU - Chakraborty, Manas

AU - Henderson, J. Nathan

AU - Hazra, Suratna

AU - Serban, Andrew J.

AU - Pham, Tuong V T

AU - Levitus, Marcia

AU - Wachter, Rebekka

PY - 2014/11/25

Y1 - 2014/11/25

N2 - We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

AB - We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

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