A bacteriophytochrome Pr/Pfr heterodimer as obtained by single-ms TR cryo-EM

Phytochromes are red/far-red light photoreceptors found across many kingdoms of life1. Upon light absorption, they reversibly interconvert between a red-absorbing Pr state and a far-red–absorbing Pfr state. Bacterial phytochromes (BphPs) regulate diverse physiological responses to red light, typically through a two-component signaling pathway6 involving a histidine kinase (HK) and a response regulator. Because the HK domain is an integral part of the BphP, its enzymatic activity is directly light-controlled. A recent cryo-EM study reported a BphP heterodimer2 consisting of one protomer in the Pr state and the other in the Pfr state, formed after exposure to ambient white laboratory light. Here, we use the Spotiton technique3,4 to deliberately generate high concentrations of the Pr/Pfr heterodimer by shortly illuminating BphP particles on a cryo-EM grid which was vitrified about 10 ms later. The resulting full-length Pr/Pfr heterodimer structure closely matches the one reported previously2. Reanalysis of earlier cryo-EM data 5 yielded a complete Pr/Pr homodimer structure with both protomers in the Pr state. Comparison of the two structures reveals that the coiled-coil linker helices connecting the HK domain to the photosensory core unwind during the transition from the homodimer to the heterodimer. The unwinding is accompanied by an almost 180° rotation of the entire HK domain. This large-scale rotation likely modulates HK activity and provides insight into the mechanism of signal perception and transduction in BphPs. Because coiled-coil helices are common in motor proteins7,8, a similar unwinding mechanism may underlie the rotational movements of their “motor heads”, impacting their dynamics and energetics as well.