FK506-binding protein 12 with the F36V mutation

Human FK506-binding protein 12 carrying the F36V substitution, which opens a cavity in the ligand-binding pocket so that a C9-bumped synthetic ligand binds FKBP F36V in preference to endogenous FKBP12. The bivalent form of that ligand, AP1903, crosslinks two FKBP F36V domains and so homodimerizes the proteins fused to them.

Length: 321 bp (107 aa)

Uses: multimerization

Cleavable: No

Origin: Human FKBP12 with F36V substitution

Characteristics

Encodes a 107-amino-acid protein of 321 nucleotides, beginning GVQVETIS and ending DVELLKLE, with Val36 in place of the Phe36 of FKBP12 and the wild-type Leu106. The F36V substitution opens a cavity of about 90 cubic angstroms that the protein does not relax to fill and into which the ethyl bump of the ligand 5S inserts, while the 1.9-angstrom crystal structure is otherwise superimposable on wild-type FKBP12. The fluorescein conjugate fluoro-5S binds FKBP F36V with a Kd of 0.094 nM against 67 nM for wild-type FKBP12, an affinity higher than that of any ligand known for wild-type FKBP12, and the bivalent AP1903 displaces a fluorescent FK506 probe from FKBP F36V with an IC50 of 5 nM while binding wild-type FKBP12 negligibly.

Applications: Chemically induced homodimerization of fused proteins by adding the bivalent ligand AP1903, which crosslinks two FKBP F36V domains. HT1080 human fibrosarcoma cells expressing a myristoylation sequence, two copies of FKBP F36V and the human Fas intracellular domain undergo apoptosis on overnight AP1903 treatment with an EC50 of about 0.1 nM, and the same two-copy Fas fusion clears cells implanted in nude mice at an intravenous AP1903 dose of 0.4 mg/kg.

Limitations: Selectivity against endogenous FKBP12 is incomplete: fluoro-5S still binds wild-type FKBP12 with a Kd of 67 nM, and in cells AP1903 discriminates an FKBP F36V Fas fusion from its wild-type counterpart by only 200-fold, against 1,000-fold in binding. FKBP F36V also requires a C9-bumped ligand, because unbumped ligands such as compound 1 bind it and wild-type FKBP12 about equally.

Sequence

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References

  1. Clackson et al. (1998). Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity. Proc Natl Acad Sci USA - Clackson 1998 FKBP F36V Bump Hole