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The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

The FKBP-derived destabilizing domains are increasingly being used to confer small molecule-dependent stability to many different proteins. The L106P domain confers instability to yellow fluorescent protein when it is fused to the N-terminus, the C-terminus, or spliced into the middle of yellow fluorescent protein, however multiple copies of L106P do not confer greater instability. These engineered destabilizing domains are not dominant to endogenous degrons that regulate protein stability.

Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.

We recently identified mutants of the human FKBP12 protein that are unstable and rapidly degraded when expressed in mammalian cells. We call these FKBP mutants destabilizing domains (DDs), because their instability is conferred to any protein fused to the DDs. A cell-permeable ligand binds tightly to the DDs and prevents their degradation, thus providing small molecule control over intracellular protein levels. We now report the synthesis and functional characterization of a stabilizing ligand called Shield-2. The synthesis of Shield-2 is efficient, and this ligand binds to the FKBP(F36V) protein with a dissociation constant of 29 nM.

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilized by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP.rapamycin.FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K(d) = 26 +/- 0.8 microM). The FKBP12.rapamycin complex, however, binds to FRB 2000-fold more tightly (K(d) = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.

Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.