Light-inducible protein heterodimerization systems, which are based on genetically encoded light-sensitive proteins and their conformation-selective binders, have emerged as powerful tools for manipulating protein activity in mammalian cells with high spatiotemporal precision. Among these, blue light-responsive pairs, such as CRY2-CIB1 [1], TULIPs [2], Magnet [3], and iLid [4], have been widely adopted due to their rapid kinetics, reversibility, and high light-to-dark switching ratios. However, their utility in deep tissues and living animals is limited by the poor penetration and phototoxicity of blue light [[5], [6]].

To overcome this constraint, red/far-red light-responsive systems have gained attention because of their deeper tissue permeability and low phototoxicity [[5], [6]]. Among currently available photoswitches, the photosensory core module (PCM) of phytochromes naturally operates within this “optical transparency window”. Their conformation exists in two stable states, a red-light-absorbing state (Pr) and a far-red-light-absorbing state (Pfr), which can be rapidly interconverted by illumination with different wavelengths of light [7]. Consequently, diverse phytochrome-based optogenetic systems have been developed. The most common class involves red/far-red light-inducible heterodimerization systems utilizing native binders, such as PhyA/FHY1 [8], PhyB/PIF [9] RpBphP1/RpPpsR2 [10] and RpBphP1/Q-PAS1 [[11], [12]], or incorporating engineered synthetic binders, such as DrBphP/LDBs [13] and MagRed [14]. In addition to heterodimerization, pioneering works also established homodimeric tools, exemplified by the iLight family [[15], [16]] and the DrBphP-based family of optically controlled receptor tyrosine kinases (RTKs) [17], [18], [19].

The recently developed system MagRed is a robust light-inducible heterodimerization tool developed by screening an affibody library to engineer a Pfr-state-selective binder for Deinococcus radiodurans bacteriophytochrome (DrBphP) [14]. The resulting binder, Aff6_V18FΔN (designated as Aff6), exhibits 20-fold higher binding affinity for DrBphP in the Pfr state than in the Pr state, with affinities in the sub-micromolar range. MagRed exhibits a high light/dark switching ratio suitable for various optogenetic applications, although notable dark-state background activity has been observed in certain contexts [20]. Additionally, MagRed utilizes biliverdin (BV), a mammalian endogenous chromophore, eliminating the need for exogenous chromophore addition. To date, MagRed has been applied in photoactivatable tetR-tetO-based gene expression [14], CRISPR-Cas9-based transcription [14], split-Cre mediated genomic recombination [14], [21], and conditional protein splicing [20].

Despite its broad potential for optogenetic applications, the structural mechanism by which Aff6 distinguishes between the Pfr and Pr conformations of DrBphP remains poorly understood. As the photoswitch module of MagRed, DrBphP-PCM is composed of a conserved homodimeric Per/Arndt/Sim-cGMP phosphodiesterase/adenyl cyclase/Fh1A-phytochrome specific (PAS-GAF-PHY) domain architecture [7]. The BV chromophore, which absorbs light in the red/far-red wavelength region, is embedded inside the GAF domain and covalently attached to a cysteine residue in the PAS domain. Upon light illumination, the fourth ring of BV undergoes photoisomerization, a key step that triggers the subsequent conformational changes in DrBphP [7]. Notably, a key “tongue” hairpin loop (residues 444–476, extending from the PHY domain to interact with the GAF domain's BV binding pocket) refolds from a β-sheet structure in the Pr state to an α-helical structure in the Pfr state, and this refolding is coupled to the opening of the dimer at the C-terminus (Fig. 1A) [[22], [23]]. Despite this general understanding of DrBphP-PCM's conformational changes, the precise binding epitope of Aff6 on DrBphP-PCM and the key molecular determinants governing its light-dependent affinity and specificity remain unresolved. High-resolution insight into this recognition event is crucial not only for understanding the molecular basis of MagRed specificity but also for guiding the rational design of improved phytochrome-based optogenetic modules.

Solution NMR spectroscopy is a versatile technique widely used in high-resolution structure determination and interaction analysis of biological macromolecules. However, NMR analysis of large proteins like DrBphP-PCM poses a significant challenge due to increased spectral complexity and reduced signal sensitivity. Two recent studies reported NMR analysis of a monomeric version of DrBphP-PCM (DrBphP-PCMmono) [[24], [25]]. They assigned a large proportion of DrBphP-PCMmono backbone resonances and analyzed its photoconversion dynamics, indicating that not only the tongue region but also the helical spine (the long α-helix connecting the PHY and GAF domain) serves as pathways through which conformational signals are transduced from BV in solution. These studies open up opportunities for detailed investigations of MagRed light-inducible heterodimerization.

In this study, we combined solution NMR spectroscopy, surface plasmon resonance (SPR), molecular docking, and mutational analysis to dissect the molecular basis of photo-state-specific Aff6 binding to DrBphP. We verify that the monomeric DrBphP-PCMmono is sufficient to mediate light-dependent heterodimerization with Aff6, and we identify the key binding region of Aff6 on DrBphP-PCMmono via NMR analysis. Guided by experimental data, we construct a computational docking model to clarify the critical interaction mediating photo-state specificity, which is further validated by mutational studies. Additionally, we identify that Aff6 exerts an allosteric effect by stabilizing the Pfr conformation of DrBphP-PCMmono, thereby retarding its Pfr-to-Pr reversion. These findings reveal the molecular mechanism of MagRed and establish a mechanistic framework for engineering next-generation phytochrome-based optogenetic tools.