Open-closed structure of light-responsive protein LOV2 regulates its molecular interaction with a binding partner

Optogenetic approaches have broad applications, including regulating cell signaling and gene expression. Photoresponsive protein LOV2 and its binding partner ZDK represent an important protein caging/uncaging optogenetic system. Herein, we combine time-resolved small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) to reveal different structural states of LOV2 and the light-controlled mechanism of interaction between LOV2 and ZDK. In response to blue light within a time frame of ca. 70 s, LOV2 has a significantly higher value of radius of gyration Rg (29.6 ± 0.3 vs 26.4 ± 0.4 Å) than its dark state, suggesting unwinding of the C-terminal Jα-helix into an open structure. Atomic force microscopy was used to characterize molecular interactions of LOV2 in open and closed states with ZDK at a single-molecule level. The closed state of LOV2 enables strong binding with ZDK, characterized by a 60-fold lower dissociation rate and a ∼1.5-times higher activation energy barrier than for its open state. In combination, these data support a light-switching mechanism that is modulated by the proximity of multiple binding sites of LOV2 for ZDK.