AN ACTIVE FK506-BINDING DOMAIN OF 17000 DALTONS IS ISOLATED FOLLOWINGLIMITED PROTEOLYSIS OF CHICKEN THYMUS HSP56

We have previously identified hsp56, a protein component of steroid re ceptor complexes, as an FK506 binding protein [Yem et al. (I 992) J. B iol. Chem. 267, 2868-2871]. We now report that hsp56 is also found to be a major immunophilin in chicken thymus, by virtue of binding to FK5 06-Affi-Gel-10 as well as positive cross-reactivity with a polyclonal antiserum directed against human hsp56. Limited digests of purified ch icken hsp56 with endoproteinase Lys C result in the production of a un ique polypeptide having a mass of about 17 kDa (p17), as judged by Wes tern blotting. Peptide mapping provided additional proof that p17 is a fragment which comprises the entire FK506 binding domain I of chicken hsp56, terminating with an Arg-Lys which might represent a processing site. Binding of radiolabeled dihydro FK506 to p17 is saturable with a calculated K(D) of 42 nM. Since size exclusion chromatography of dru g-p17 complexes indicates that the active species is a homodimer with a mass of 30-40 kDa, the stoichiometry calculated for the drug-protein complex is approximately 1:1. Furthermore, unlike FKBP-12, chicken p1 7 bound to FK506 does not bind to calcineurin-calmodulin complexes. Th is work demonstrates the excision of a domain from an hsp56 protein th at is active in binding FK506 and functionally distinct from FKBP-12, a protein of similar size and structure.