Structure and Function Studies of FKBP65:A Putative Molecular Chaperone of Tropoelastin

Abstract

FKBP-65 is a member of the immunophilin class of proteins consisting primarily of the cyclophilins and the FKBP's which bind the immunosuppressant drugs cyclosporin A and FK506, respectively. Immunophilins possess peptidylprolyl cis-trans isomerase (PPiase) activity which is inhibited upon binding of their respective macrolides. Specific cellular targets of most immunophilins and the role of PPiase activity in vivo remain largely unknown. FKBP-65 has been proposed as a molecular chaperone of tropoelastin (TE), the soluble precursor of elastin (Davis et al. 1998). TE contains 12% proline residues, many of which are found in VPGVG repeats. When P2 is in the trans conformation, these motifs form repeated type-II ~-turns and ~-spirals resulting in selfassociation of TE via an inverse temperature-dependent transition known as coacervation. Coacervation can be monitored by turbidity increases at 300 nm. We have used purified recombinant FKBP-65 in coacervation assays with chick aorta TE to show that FKBP65 specifically affects the coacervation characteristics of TE in a concentration-dependant manner. The overall extent of coacervation of TE could be increased by more than 2-fold over controls by inclusion of nM amounts of FKBP-65 in the assay. Also, FKBP-65 decreases the coacervation onset temperature of TE by 5-l 0°C. Structural evidence suggests that the influence of FKBP65 on tropoelastin coacervation may be due to its ability to increase the ~ structural content of tropoelastin. These results suggest that FKBP-65 may be a physiologically relevant, TE-specific molecular chaperone.