A ligand-reversible dimerization system for controlling protein-protein interactions

Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains a re fused to a signaling domain of interest, allowing crosslinking to be ini tiated by addition of a bivalent FKBP ligand. In the course of protein engi neering studies on human FKBP, we discovered that a single point mutation i n the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analyti cal ultracentrifugation, and x-ray crystallographic studies show that the m utant (FM) forms discrete homodimers with micromolar affinity that can be c ompletely dissociated within minutes by addition of monomeric synthetic lig ands. These unexpected properties form the basis for a "reverse dimerizatio n" regulatory system involving FM fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have use d this strategy to rapidly and reversibly aggregate fusion proteins in diff erent cellular compartments, and to provide an off switch for transcription . Reiterated FM domains should be generally useful as conditional aggregati on domains (CADs) to control intracellular events where rapid, reversible d issolution of interactions is required. Our results also suggest that dimer ization is a latent property of the FKBP fold: the crystal structure reveal s a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dram atic change in quaternary structure.