Enhancing response of a protein conformational switch by using two disordered ligand binding domains

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Synthetic Biology

Background:

• Protein conformational switches typically use one input domain to control an output domain's biological response.
• Harnessing energy from two binding domains to drive a single output domain's conformational change presents a design challenge.
• Previous designs relied on binding-induced folding of a single input domain.

Purpose of the Study:

• To engineer a novel protein switch capable of integrating signals from two input domains.
• To achieve synergistic conformational changes in an output domain using dual binding events.
• To create a switchable enzyme with tunable responsiveness based on ligand concentration.

Main Methods:

• Engineered barnase (Bn) into a switchable enzyme by creating native (OFF) and circularly permuted (ON) folds.
• Inserted two unstable FK506 binding protein (FKBP) variants into engineered barnase junctions.
• Utilized rapamycin to induce folding of FKBP domains, triggering conformational changes in barnase.

Main Results:

• Rapamycin-induced FKBP folding unfolded the native barnase fold via mutually exclusive folding.
• Rapamycin-induced permuted FKBP folding stabilized the permuted barnase fold through loop-closure entropy.
• These complementary folding events synergistically activated ribonuclease (RNase) function, validated in yeast, human cells, and in vitro.

Conclusions:

• Demonstrated a novel protein switch design integrating dual input domains for precise biological control.
• The switch operates via a combination of loop-closure entropy and mutually exclusive folding, analogous to a mechanical transmission.
• This system offers efficient switching at low ligand concentrations and maximal response at high concentrations.