Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6.

Hui Chen,David M LeMaster,Griselda Hernández,Hongmin Li,Annie Héroux,Sourajit M Mustafi,Zhong Li

doi:10.1107/s1399004713032112

Abstract

The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca(2+) channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2₁ and P3₁21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3₁21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in the P2₁ crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

Highlights

The human genome encodes two homologous 12 kDa FK506binding proteins, FKBP12 and FKBP12.6, which differ at 18 residue positions (Supplementary Fig. S11)
The resultant occlusion of the standard ligand-binding site were argued to be driven by compression forces that impinge upon this aromatic ring (Szep et al, 2009)
In the P3121 crystal form of unligated FKBP12.6, a similar perpendicular reorientation of the Phe59 ring is observed. In this case the absence of substantial steric interactions appears to allow the facile sampling of the two orientations of the Phe59 ring in the same crystal form. This enhanced plasticity in the active site of FKBP12.6 is likely to contribute to the marked attenuation in the spatial extent of the residues that exhibit doubling of their amide resonances compared with those of the homologous FKBP12

Summary

Introduction

The human genome encodes two homologous 12 kDa FK506binding proteins, FKBP12 and FKBP12.6, which differ at 18 residue positions (Supplementary Fig. S11) Each of these proteins can bind the immunosuppressant FK506 to form a ternary complex with the protein phosphatase calcineurin which, by inhibiting the activity of this enzyme, blocks a key T-cell activation pathway involved in tissue transplant rejection (Liu et al, 1991). Both FKBP12 and FKBP12.6 can bind the immunosuppressant rapamycin to form an inhibitory ternary complex with the protein kinase mTOR (mammalian target of rapamycin), which plays a major role in regulating cell growth and cancer progression (Heitman et al, 1991). Only a single X-ray structure of FKBP12.6 has been deposited in the Protein Data Bank, in which the protein is bound to rapamycin (Deivanayagam et al, 2000)

Methods

Results

Conclusion