An active FK506-binding domain of 17,000 daltons is isolated following limited proteolysis of chicken thymus hsp56.
We have previously identified hsp56, a protein component of steroid receptor complexes, as an FK506 binding protein [Yem et al. (1992) J. Biol. 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 FK506-Affi-Gel-10 as well as positive cross-reactivity with a polyclonal antiserum directed against human hsp56. Limited digests of purified chicken hsp56 with endoproteinase Lys C result in the production of a unique polypeptide having a mass of about 17 kDa (p17), as judged by Western 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 KD of 42 nM. Since size exclusion chromatography of drug-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 p17 bound to FK506 does not bind to calcineurin-calmodulin complexes. This work demonstrates the excision of a domain from an hsp56 protein that is active in binding FK506 and functionally distinct from FKBP-12, a protein of similar size and structure.
Yem
A W
AW
Department of Biochemistry, Upjohn Company, Kalamazoo, Michigan 49001.
Reardon
I M
IM
Leone
J W
JW
Heinrikson
R L
RL
Deibel
M R
MR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D010450:Endopeptidases / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins; D013950:Thymus Gland / Q000737:chemistry*
D000596:Amino Acids; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D010450:Endopeptidases; C044574:lysine-C peptidase; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus