title: Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.
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.
authors.affiliation: Department of Biochemistry & Biophysics.
authors.forename: Gurkan
authors.initials: G
authors.lastname: Guntas
authors.affiliation: Department of Biochemistry & Biophysics.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Hallett
authors.affiliation: Department of Biochemistry & Biophysics.
authors.forename: Seth P
authors.initials: SP
authors.lastname: Zimmerman
authors.affiliation: Department of Biochemistry & Biophysics.
authors.forename: Tishan
authors.initials: T
authors.lastname: Williams
authors.affiliation: Department of Biochemistry & Biophysics.
authors.forename: Hayretin
authors.initials: H
authors.lastname: Yumerefendi
authors.affiliation: Department of Cell Biology & Physiology, University of North Carolina Lineberger Comprehensive Cancer Center, and Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: James E
authors.initials: JE
authors.lastname: Bear
authors.affiliation: Department of Biochemistry & Biophysics, University of North Carolina Lineberger Comprehensive Cancer Center, and bkuhlman@email.unc.edu.
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
mesh_terms: D000595:Amino Acid Sequence; D018554:Avena; D061505:Cell Surface Display Techniques; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D055503:Protein Multimerization / Q000528:radiation effects*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000528:radiation effects; D015398:Signal Transduction / Q000528:radiation effects*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D050505:Mutant Proteins; D010940:Plant Proteins; D020558:GTP Phosphohydrolases
keywords: PER-ARNT-SIM domain; Rosetta molecular modeling suite; computational library; optogenetic tool; phage display