Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamics-driven allostery is key to understanding signal integration on a molecular level. We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a non-photochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like fold-changes of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox.
Keywordsphotoreceptor
photocycle
signal integration
HDX-MS
c-di-GMP
AbbreviationsAtPhyBArabidopsis thaliana phytochrome B
HDX-MShydrogen-deuterium exchange coupled with mass spectrometry
HPLChigh-performance liquid chromatography
IsPadCIdiomarina species A28L phytochrome-activated diguanylate cyclase
PaBphPPseudomonas aeruginosa bacteriophytochrome
XccBphPXanthomonas campestris bacteriophytochrome
© 2024 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
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