Structural insights into photo-state-specific binding of affibody Aff6 to the photosensory core module of DrBphP

Light-inducible heterodimerization systems offer precise, reversible control of protein interactions in living cells. Leveraging the high tissue-penetration of red/far-red light, the MagRed system, composed of a bacteriophytochrome Deinococcus radiodurans BphP (DrBphP) and its engineered affibody binder Aff6, achieves robust photoswitchable dimerization. This makes MagRed well-suited for in vivo and deep-tissue optogenetic application. However, the structural mechanism underlying Aff6's photo-state-specific recognition of DrBphP remains elusive. Here, we combine solution NMR spectroscopy, surface plasmon resonance (SPR), molecular docking and mutational analysis to elucidate the light-dependent interaction between a monomeric photosensory core module of DrBphP (DrBphP-PCM_mono) and Aff6. We show that DrBphP-PCM_mono alone is sufficient for light-inducible heterodimerization with Aff6, exhibiting a ∼ 23-fold affinity difference between the Pfr and Pr states. NMR titration reveals that Aff6 binds primarily to the PHY domain and the C-terminal region of the helical spine. Furthermore, docking and mutagenesis identify a key aromatic interaction (involving F327/H334 of DrBphP and F18 of Aff6) as the molecular basis for this conformational selectivity. Additionally, Aff6 binding stabilizes the Pfr state and retards the Pfr-to-Pr reversion of DrBphP-PCM_mono. These findings not only provide critical structural insight into MagRed function but also establish a foundation for rationally engineering next-generation phytochrome-based optogenetic tools.