Crystal structures of a far-red photoreceptor in different light-absorbing states: insights into spectral tuning and light signaling

Abstract

Cyanobacteriochromes (CBCRs) are bilin-binding photoreceptors with remarkable spectral versatility. Using phycocyanobilin (PCB) as a chromophore, CBCRs regulate diverse light-dependent processes in cyanobacteria, ranging from photosynthesis to chromatic acclimation. Although extensive studies have uncovered multiple spectral tuning mechanisms in bilin-binding proteins, recent structural studies of far-red CBCRs suggest the existence of additional tuning strategies in both the 15Z and 15E states. Here we report crystal structures of the representative far-red CBCR Anacy_2551g3 in three distinct light-absorbing states, all of which adopt a compact all-syn PCB conformation. These structures demonstrate that 15Z/15E photoisomerization in Anacy_2551g3 involves minimal chromophore rotation relative to the GAF domain, in stark contrast to other characterized bilin-based photoreceptors. To investigate the molecular basis of its far-red absorption, we examined the protonation and tautomeric states of PCB in the Pfr state using resonance Raman (RR) spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Comparisons of experimental and calculated RR spectra support a bilin lactam as the predominant tautomeric form in the Pfr state. Integrating structural, spectroscopic, computational and mutational analyses, we propose that specific protein-chromophore interactions play critical roles in modulating chromophore conjugation beyond bilin coplanarity. Structural analyses further suggest a signaling model in which light regulation by Anacy_2551g3 is mediated through reversible switching between a high-affinity Pfr state and a low-affinity Po state that does not involve large chromophore motions. Together, these results provide new insights into how protein-chromophore coupling governs spectral tuning and light signaling in bilin-based photoreceptors. Significance statementBilins are widespread biological pigments that mediate photoreception, light harvesting, and photosynthesis across diverse light environments. In a phenomenon known as spectral tuning, the optical properties of bilin-binding proteins are profoundly influenced by protein-chromophore interactions. Mechanistic understanding of spectral tuning and light signaling is important not only for advancing fundamental knowledge of light-sensitive proteins but also for developing new engineering strategies in synthetic biology and biotechnology. Recently discovered cyanobacteriochromes (CBCRs) exhibit remarkable spectral diversity and structural versatility, providing excellent model systems for dissecting the mechanisms of bilin-based photoreceptors. By integrating crystallography, spectroscopy and computational methods, this work examines three distinct light absorbing states of a representative far-red CBCR. Our findings reveal previously unrecognized mechanisms of spectral tuning and light signaling, highlighting the critical roles of protein-chromophore coupling and electrostatic interactions in regulating photoreceptor function.