A Bioorthogonal Small‐Molecule‐Switch System for Controlling Protein Function in Live Cells

Abstract

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP' (SLF') linked to trimethoprim (TMP). The SLF' moiety binds to the F36V mutant of FK506-binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF'-TMP-induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM. Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.

Synthetic mechanobiology: engineering cellular force generation and signaling

“…Various reversible CID systems have been reported[28,29]. Alternatively, a second, orthogonal dimerizing agent can be used to relocate a rapamycin-induced FRB-FKBP complex from the plasma membrane to mitochondrial membranes to produce a pulse of localized protein activation[30,31].…”

Multidirectional Activity Control of Cellular Processes by a Versatile Chemo‐optogenetic Approach

“…NvocTMP-Cl can covalently pre-localize to aprotein fused with aHaloTag. [10] The NvocTMP-Cl and S*T/ST-induced dimerization is stable and essentially irreversible even after wash-out, but can be disrupted by the competitor TMP to make both systems reversible (Supporting Information, Figures S1-S3). On the other hand, SLF*-TMP and SLF'-TMP feature as ynthetic ligand of FKBP-(F36V) (SLF')a nd aT MP moiety.S LF*-TMP (S*T) is an optically pure diastereomer while SLF'-TMP (ST) is ad iastereomeric mixture.S LF*/SLF' binds with high affinity (subnanomolar) to the FKBP(F36V) mutant (FKBP')w ith a1 000-fold selectivity over wild-type FKBP.B oth dimerizers induce dimerization between FKBP'-a nd eDHFRfused proteins with nanomolar affinity (Supporting Information, Figure S1E,F).…”

“…This system combines either the newly introduced secondgeneration SLF*-TMP (S*T) dimerizer or the first-generation SLF'-TMP (ST) [10] with the NvocTMP-Cl photocaged dimerizer, [11] which were recently established in our lab ( Figure 1; Supporting Information, Figure S1). NvocTMP-Cl consists of caged trimethoprim (TMP), aH aloTag ligand (chlorohexyl group) and ap olyethylene glycol (PEG) linker.T MP selectively binds to Escherichia coli dihydrofolate reductase (eDHFR) with a K d of 1nm and the chlorohexyl moiety can form ac ovalent bond with ab acterial alkyl dehalogenase mutant (HaloTag).…”

“…On the other hand, SLF*-TMP and SLF'-TMP feature as ynthetic ligand of FKBP-(F36V) (SLF')a nd aT MP moiety.S LF*-TMP (S*T) is an optically pure diastereomer while SLF'-TMP (ST) is ad iastereomeric mixture.S LF*/SLF' binds with high affinity (subnanomolar) to the FKBP(F36V) mutant (FKBP')w ith a1 000-fold selectivity over wild-type FKBP.B oth dimerizers induce dimerization between FKBP'-a nd eDHFRfused proteins with nanomolar affinity (Supporting Information, Figure S1E,F). [10] The NvocTMP-Cl and S*T/ST-induced dimerization is stable and essentially irreversible even after wash-out, but can be disrupted by the competitor TMP to make both systems reversible (Supporting Information, Figures S1-S3). TheN vocTMP-Cl system is tunable with doses of illumination (Supporting Information, Figure S4) and the induced dimerization is very rapid (t 1/2 = 0.55-1.69 s; Supporting Information, Figure S5).…”

Optical control over monomeric and multimeric protein hybrids

“…Such processes can be reversed for instance by adding a competitor binding. [46,53] An option for reversible optical dimerizers is already exemplified by Nature in the form of blue light-responsive LOV system. As elaborated in the previous section on allosteric regulation, in some examples such as in algal aureochrome, the photo-switching of LOV activates homodimerization that further enables binding event.…”