Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes

Abstract

Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the common target for two immunophilin-immunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FKBP-FK506. How the two structurally distinct immunophilin-drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-A resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike FKBP-FK506, CyPA-CsA interacts with Arg-122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. The simultaneous interaction of CyPA with both the composite surface and the active site of CN suggests that the composite surface may serve as a substrate recognition site responsible for the narrow substrate specificity of CN. The comparison of CyPA-CsA-CN with FKBP-FK506-CN significantly contributes to understanding the molecular basis of regulation of CN activity by the immunophilin-immunosuppressant.

Fig 1.

Stereoview of electron density for CsA. The (2F_o − F_c) map was calculated from the structure-omitted CsA and contoured at 1.5σ.

Fig 2.

Ribbon representation of CyPA-CsA-CN. Color codes are CNA, gold; CNB, cyan; CsA, green; CyPA, red; Zn²⁺ and Fe³⁺, pink; and calcium, blue. The residues from CN involved in binding of CyPA-CsA are shown as blue balls.

Fig 3.

Stereoview of the superposition of C^α atoms of CyPA-CsA-CN (red) over unligated CNA (green) and FKBP-FK506-CN (yellow). The complex structures were overlaid with optimal overlap of the catalytic domain of CNA.

Fig 4.

Interfacial interactions between CN and immunophilins–immunosuppressants. CNA and CNB are shown as yellow and cyan ribbons. (A) The CN composite surface for CyPA-CsA binding. A total of 25 residues of CN are involved in interaction with CyPA-CsA: Arg-122, Tyr-159, Phe-160, Leu-312, Val-314, Tyr-315, Tyr-341, Trp-342, Pro-344, Asn-345, Trp-352, Ser-353, Pro-355, Phe-356, and Glu-359 of CNA, and Glu-47, Gln-50, Met-118, Val-119, Asn-122, Leu-123, Lys-124, and Lys-164 of CNB. Red balls represent residues interacting with CyPA and green balls represent residues interacting with CsA (green sticks). The residues from CNB are marked with the letter “B” attached to the residue number. (B) The CN surface for binding of CyPA-CsA and FKBP12-FK506. CN residues in red interact with both CyPA-CsA and FKBP12-FK506. Green residues are unique for CyPA-CsA binding and blue residues are unique for FKBP-FK506. CsA and FK506 are shown as green and blue sticks. (C) CyPA residues for recognition of CN (gold) and CsA (blue). CsA is shown in green sticks.

Fig 5.

Superposition of CsA in the binary CyPA-CsA complex (purple), CsA in the ternary CyPA-CsA-CN complex (gold), and MeBm₂t-CsA in the binary CyPA-MeBm₂tCsA complex (blue). MeBmt, N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine; MeBm₂t, N-methyl-4-[(E)-2-butenyl]-4,4-dimethyl-l-threonine. Color codes for atoms: carbons as green balls, oxygen as red, and nitrogen as blue. Residues of MeLeu-9 to 2-aminobutyrate (Abu)-2 (top) bind CyPA, whereas MeLeu-4 to Ala-7 bind CN (bottom).