Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

Abstract

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this longstanding conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics. radiodurans (DrBphP), light induces extensive structural changes in the photosensory module that are relayed to the output module 29 .In terms of enzymatic activity, the dark-adapted Pr state exhibited higher kinase activity than the Pfr state in cyanobacterial phytochrome Cph1, 27,30,31 , similar to other bacteriophytochromes 32 . In particular, the bacteriophytochrome from Agrobacterium fabrum (AgP1) displays histidine kinase activity in its resting Pr state 28,33 ; in the Pfr state, the autophosphorylation and phosphotransfer reactions are down-regulated by 2-fold and 10-fold, respectively 28 . This kinase activity of the AgP1 has been shown to control bacterial conjugation 34 . By contrast, no kinase activity has been demonstrated for DrBphP, notwithstanding close sequence homology and the elaborate structural changes this receptor undergoes under light 22,29 . Despite the eminent role of DrBphP as a paradigm for photoreception, the enzymatic activity and the physiological role of this model phytochrome have hence remained enigmatic.Here, we unravel this long-standing puzzle by studying the enzymatic activity and interactions of DrBphP and AgP1, as two canonical bacteriophytochromes with HK effector domains. By pursuing an integrated biochemical and structural strategy, we show that despite close homology, AgP1 acts as a histidine kinase whereas DrBphP functions as a light-activated phosphatase. Our biochemical and structural data pinpoint two key residues proximal to the catalytic histidine that affect the balance between the kinase and phosphatase activities. Together, the two phytochromes provide soluble, lightcontrollable systems with opposite activities for the study and application of two-component signaling.

Cryo-Electron Microscopy of Arabidopsis thaliana Phytochrome A in Its Pr State Reveals Head-to-Head Homodimeric Architecture

“…We tentatively ascribed these density differences to varying degrees of static and dynamic structural heterogeneity in the PCM and CTM. In support of this notion, we note that sensor histidine kinases, and by that token the HKRD in AtphyA, exhibit inherently high levels of structural malleability that underpins their function (Gushchin and Gordeliy, 2018;Buschiazzo and Trajtenberg, 2019;Möglich, 2019;Multamäki et al, 2020).…”

Lack of the Bacterial Phytochrome Protein Decreases Deinococcus radiodurans Resistance to Mitomycin C

“…DrBphP binds BV via a conserved cysteine (Cys-24) upstream of the PAS domain (Wagner et al, 2005). The DrBphP histidine residue, His-532, is one of the three central DrBphP residues that interact with DrBphR (Multamäki et al, 2020). We constructed DrBphP derivatives using alanine substitution at amino acids Cys-24 and His-532.…”

“…Deinococcus radiodurans BphP dephosphorylates its cognate response regulator (RR), DrBphR (Multamäki et al, 2020).…”

“…In D. radiodurans, the bphP gene immediately precedes the bphR gene, which encodes a putative cognate RR of DrBphP, named DrBphR (Figure 1A). However, a recent study showed that DrBphP lacks autokinase or phosphotransfer activities in both the Pr and Pfr states; thus it does not produce phosphorylated DrBphR (phospho-DrBphR; Multamäki et al, 2020). Instead, DrBphP functions exclusively as a light-activated phosphatase.…”

“…Instead, DrBphP functions exclusively as a light-activated phosphatase. DrBphP can dephosphorylate phospho-DrBphR, which is phosphorylated using a non-enzymatic mechanism or by other HKs (Multamäki et al, 2020).…”