Download MendeleyEngineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.
Guntas, G., Hallett, R.A., Zimmerman, S.P., Williams, T., Yumerefendi, H., Bear, J.E., Kuhlman, B.(2015) Proc Natl Acad Sci U S A 112: 112-117
- PubMed: 25535392
- DOI: https://doi.org/10.1073/pnas.1417910112
- Primary Citation of Related Structures:
4WF0 - PubMed Abstract:
The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
Organizational Affiliation:
Department of Biochemistry & Biophysics.
