4WF0

Crystal Structure of iLID - an Improved Light-Inducible Dimer

Summary for 4WF0

• Entry DOI: 10.2210/pdb4wf0/pdb
• Descriptor: NPH1-1, FLAVIN MONONUCLEOTIDE, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: light-inducible dimer, flavoprotein
• Biological source: Avena sativa (Oat)
• Total number of polymer chains: 2
• Total formula weight: 37523.27

Authors

Hallett, R.,Williams, T.,Kuhlman, B. (deposition date: 2014-09-11, release date: 2014-12-24, Last modification date: 2023-09-27)

Primary citation

Guntas, G.,Hallett, R.A.,Zimmerman, S.P.,Williams, T.,Yumerefendi, H.,Bear, J.E.,Kuhlman, B.
Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.
Proc.Natl.Acad.Sci.USA, 112:112-117, 2015

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.

PubMed: 25535392
DOI: 10.1073/pnas.1417910112

Experimental method

X-RAY DIFFRACTION (1.95 Å)