Calcineurin in a Crowded World

Abstract

Calcineurin is a Ser/Thr phosphatase that is important for key biological processes, including immune system activation. We previously identified a region in the intrinsically disordered regulatory domain of calcineurin that forms a critical amphipathic α-helix (the "distal helix") that is required for complete activation of calcineurin. This distal helix was shown to have a Tm close to that of human body temperature. Because the Tm was determined in dilute buffer, we hypothesized that other factors inherent to a cellular environment might modulate the stability of the distal helix. One such factor that contributes to stability in other proteins is macromolecular crowding. The cell cytoplasm is comprised of up to 400 g/L protein, lipids, nucleic acids, and other compounds. We hypothesize that the presence of such crowders could increase the thermal stability of the distal helix and thus lead to a more robust activation of calcineurin in vivo. Using biophysical and biochemical approaches, we show that the distal helix of calcineurin is indeed stabilized when crowded by the synthetic polymers dextran 70 and ficoll 70, and that this stabilization of the distal helix increases the activity of calcineurin.

Figure 1

Activation of calcineurin by calmodulin. (A) The CaM-binding site is located on the regulatory domain (RD), which interacts with the A chain–B chain interface in the absence of Ca²⁺. (B) The RD is released when the B chain of CaN binds Ca²⁺. (C) When Ca²⁺/CaM binds to the CaM-binding site of CaN, the distal helix folds onto CaM, and the AID dissociates from the active site, fully activating the phosphatase.

Figure 2

Structural effects of dextran 70 and ficoll 70 on the RD:CaM complex using far-UV CD. RD construct in the presence and absence of CaM at various concentrations of (A) dextran 70 and (B) ficoll 70 (0–200 g/L). (C) Molar ellipticity at 222 nm of RD with CaM as a function of dextran 70 or ficoll 70 concentration showing similar degrees of crowding agent-induced structural content increase.

Figure 3

Far-UV CD spectra with small molecular osmolytes. (A) RD construct in the presence of CaM and buffer alone, 200 g/L dextrose, 200 g/L sucrose, and 40% TFE. (B) pCaN:CaM complex in 200 g/L dextran 70, 200 g/L sucrose, and 40% TFE compared to that in buffer alone.

Figure 4

CD temperature scans at 222 nm. (A) RD:CaM complex in the presence and absence of either 200 g/L dextran 70 or 200 g/L ficoll 70 with first derivatives shown as dashed lines of the same color. (B) RD construct alone in the presence and absence of 200 g/L dextran 70 or 200 g/L ficoll 70. (C) pCaN:CaM complex in the presence and absence of 8 M urea.

Figure 5

Enzyme kinetic data showing the rate of hydrolysis of pNPP as a function of pNPP concentration at 37 °C. (A) Dextran 70 and ficoll 70 increase the activity of CaN. (B) The small molecules dextrose and sucrose do not alter kinetics. (C) Truncation of CaN and removal of the AID and CT domains and its activity with ficoll 70 or dextran 70 compared to dilute buffer.