Structural basis for activation of calcineurin by calmodulin

Abstract

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

Figure 1

Structure and activation of human αCaN. a. Domain structure of the αCaN A chain highlighting the regulatory domain, with CaM binding region, and autoinhibitory domain. b. Model for CaN activation by CaM.

Figure 2

Sequence of the αCaN RD-AID-CT construct used in this work. Residues in gray were added to aid with expression and purification. The RD is denoted by green residues, with the CaM binding region highlighted in orange. The AID sequence is shown in red and the CT in black. Residue numbering is based on that for the full-length αCaNA chain.

Figure 3

PONDR prediction of αCaNA. The catalytic domain is blue, RD green, CaM binding region orange, AID red and CT dark gray.

Figure 4

Fluorescence emission spectra for wild type αCaN and four mutants with introduced tryptophan residues in the a. absence and b. presence of CaM.

Figure 5

Fluorescence anisotropy results for a fluorescently labeled RD-AID-CT construct in the presence of a two-fold excess CaM, the truncated αCaN373stop, and both.

Figure 6

Experimental evidence for the disordered nature of the RD-AID-CT and its acquisition of α-helical structure upon CaM binding. a. CD spectra for the RD-AID-CT, CaM, the CaM:RD-AID-CT complex, and a complex of CaM bound to pCaN (the CaM binding domain from αCaN). b. HXMS data for the intact RD-AID-CT in the absence and presence of CaM. The data shown are from single experiments and are not corrected for back-exchange.

Figure 7

SDS-PAGE gel showing the results of a tryptic digest of the RD-AID-CT construct in the presence of a ten-fold excess of CaM.

Figure 8

HXMS heat map for the RD-AID-CT in the absence and presence of a two-fold excess of CaM. Structure of the RD-AID-CT construct is shown at top for reference. The map was constructed using a minimal set of ten non-overlapping peptides for which complete duplicate data were available. See Figure S2 for complete deuterium uptake kinetics for all RD-AID-CT peptides.