ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

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.

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

“…Surprisingly, cotton ␤-Rca was shown to turn over five to six times more slowly than tobacco ␤-Rca, with an estimated turnover rate of 3.8 Ϯ 1.2 min Ϫ1 (n ϭ 6) under substrate-saturation conditions (28). In these preliminary experiments, we have estimated a K m value for ATP hydrolysis of about 1.5 mM, suggesting substantially weaker substrate binding compared with tobacco ␤-Rca.…”

“…2a). In this way, the K i value for ADP was extracted to be 37 Ϯ 7.0 M (Table 3), about one-third of the K m value for ATP, raising the possibility of tighter product binding compared with substrate and reflecting previously reported thermal stability data (28).…”

“…Using the NADH assay (52), no residual ADP was detected in apo-Rca preparations. However, trace amounts of ADP are introduced when adding ATP to initiate turnover, as indicated by our measurements of 4 M ADP contamination in 1 mM ATP (28).…”

“…In general, higher plant Rca preparations are highly polydisperse in vitro. Recently, we have provided experimental evidence that in vitro the hexamer formation of cotton ␤-Rca is maximized around 30 M subunit concentration (27), with close to 80% of protomers forming six-subunit assemblies in the presence of excess ATP␥S and high magnesium ion concentrations (28). These data suggest that in the chloroplast stroma, Rca is always fully assembled to a hexameric or higher order form.…”

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop

“…We therefore produced an Asp-173-Ala mutant of OsRcab. Asp-173 is the conserved Walker B acidic residue and when mutated, leads to loss of ATPase activity and nucleotide binding (van de Loo and Salvucci, 1998;Kuriata et al, 2014;Hasse et al, 2015;Fig. 3B).…”