Rational Design of a Photoactivatable Cofilin Analog using a Novel Lov-Binding Protein

The spatial and temporal organization of protein activity is an essential but poorly understood aspect of cellular signaling. Optogenetics is emerging as a powerful tool for precise modulation of specific protein's activity within living cells and animals. For non-channel proteins, optical control of protein activity has been accomplished through light-regulated protein dimerization, oligomerization, and by light-controlled steric block of a protein or peptide active site using the LOV (light-oxygen-voltage) domain. Illumination triggers a conformational change in LOV that reversibly releases the steric block. A challenge in this approach has been the difficulty of controlling the orientation of LOV in the dark to efficiently block the protein site. Recently our lab has generated a novel LOV-binding protein based on z-affibodies, a class of engineered protein scaffold. Our z-affibody (termed Zdk) binds selectively to the dark state of LOV ( 4 μM affinity in the light). We show that Zdk can be used to control the orientation of LOV in the dark state and efficiently block the actin-binding site of cofilin in a light-dependent, reversible manner. To make photoactivatable cofilin (PA-cofilin), Zdk was fused to the N-terminus of cofilin and LOV was fused to the C-terminus. We used Rosetta-based molecular modeling to rationally design linkers that oriented the Zdk-LOV complex over the actin-binding interface and also to rationally design Zdk variants with a range of affinities for LOV. Using a Zdk of the optimum affinity for LOV was found to be critical in the design. PA-cofilin undergoes a five-fold increase in actin-binding in response to light, demonstrating the tight control of protein activity that can be achieved using Zdk. Ongoing studies using PA-cofilin to probe the role of cofilin in polarized migration will be described.