Chemical mechanism of allosteric and asymmetric dark reversion in a bacterial phytochrome uncovered by cryo-EM.

Abstract

Phytochromes are light-sensitive proteins found in plants, fungi, and bacteria. They exist in two functional states, Pr and Pfr, distinguished by Z/E isomers of their bilin chromophore. The chromophore can photoswitch between these states, but also thermally converts in darkness. Despite the importance of the latter reaction, it remains unclear how it is controlled by the phytochrome. Here, we present single-particle cryo-EM measurements on the Pseudomonas aeruginosa bacteriophytochrome (PaBphP) carried out at multiple time points during dark reversion from Pr to Pfr. These experiments resolve the structure of a PrPfr hybrid state. Surprisingly, we find that only protomer B converts back to Pfr in the hybrid, while protomer A remains in Pr. We identify structural asymmetries in the precursor Pr state, which extend from the homodimer interface to a conserved histidine (H277). The hydrogen-bonding network around the chromophore is modulated, explaining how the phytochrome gains control over the activation energy of the isomerization reaction. These findings establish that dark reversion is governed by conformational selection between two substates, whereby one is "dark-reversion ready" and the other one blocks the reaction. Moreover, we explain how the equilibrium of the states is allosterically controlled across the dimer. Together, these findings provide a structural framework for tuning phytochrome signaling lifetimes in optogenetic applications. Significance statementThe dark reversion reaction of phytochromes is crucial to their signalling role in plants, bacteria, and fungi, but it is vastly understudied in terms of its chemical mechanism. It remains unsolved how the reaction can proceed at all, given that the activation energy is prohibitively high for the isomerization to occur in solution. Using time-resolved cryo-EM, we present a chemical and structural framework for understanding how the protein binding pocket regulates the reaction. Our results establish that conformational selection between two substates controls dark reversion, providing a rare example of strongly asymmetric reactivity across a dimeric protein. This opens the way for rational engineering of the lifetimes of the signaling states in phytochromes.