Circadian oscillator proteins across the kingdoms of life : Structural aspects 06 Biological Sciences 0601 Biochemistry and Cell Biology

Circadian oscillators are networks of biochemical feedback loops that generate 24-hour rhythms and control numerous biological processes in a range of organisms. These periodic rhythms are the result of a complex interplay of interactions among clock components. These components are specific to the organism but share molecular mechanisms that are similar across kingdoms. The elucidation of clock mechanisms in different kingdoms has recently started to attain the level of structural interpretation. A full understanding of these molecular processes requires detailed knowledge, not only of the biochemical and biophysical properties of clock proteins and their interactions, but also the three-dimensional structure of clockwork components. Posttranslational modifications (such as phosphorylation) and protein-protein interactions, have become a central focus of recent research, in particular the complex interactions mediated by the phosphorylation of clock proteins and the formation of multimeric protein complexes that regulate clock genes at transcriptional and translational levels. The three-dimensional structures for the cyanobacterial clock components are well understood, and progress is underway to comprehend the mechanistic details. However, structural recognition of the eukaryotic clock has just begun. This review serves as a primer as the clock communities move towards the exciting realm of structural biology

The phocycle of a flavin-binding domain of the blue-light photoreceptor phototropin

The plant blue light receptor, phot1, a member of the phototropin family (1), is a plasma membrane-associated flavoprotein that contains two (∼110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. <i>E. coli</i>-expressed LOV domains bind riboflavin-5′-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient ∼660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-μs time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of ∼50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys<sup>39</sup> thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys<sup>39</sup> exists as a thiolate

Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV<sub>390</sub>) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV<sub>390</sub> formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile → Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C−S cysteinyl adduct