Changes in active site histidine hydrogen bonding trigger cryptochrome activation

Abstract

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pK a . Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.light sensing | flavoprotein | photochemistry | redox | molecular dynamics C ryptochromes (CRYs) are flavin-binding proteins that perform a variety of sensory and catalytic functions in all kingdoms of life (1, 2). CRYs are closely related to the DNA photolyases (PLs), which catalyze light-driven redox reactions to break apart pyrimidine dimers in UV-damaged DNA (1, 2). CRYs and PLs share a conserved photolyase homology region that consists of an α-helical domain, which binds flavin adenine dinucleotide (FAD) and an α/β Rossman-fold domain, which sometimes binds a pteridine or deazaflavin antenna cofactor. CRYs also contain C-terminal extensions of variable sizes that contribute to their specific functions. The range of activities found for CRYs and PLs require that their flavin cofactors assume a broad range of redox, protonation, and excited states (1, 2).In the fruit fly Drosophila melanogaster, a type I cryptochrome (dCRY) is the primary light receptor of the circadian clock (1, 3). In response to blue light, dCRY coordinates interactions between Timeless (TIM) and the E3-ubiquitin ligase Jetlag (JET) (4). JETmediated proteolysis of TIM destabilizes its partner Period (PER). PER serves as the principal repressor of circadian gene expression and its degradation phase-shifts the clock (3). dCRY also catalyzes light-induced self-degradation that involves another E3-ligase: Brwd3 or RAMSHACKLE (5)...

Molecular dynamics simulations disclose early stages of the photo-activation of cryptochrome 4

“…the first, structure from the ensemble was used to initiate the method. Following [45], in the molecular alignment step only residues with a root mean square fluctuation of less than 1.3 Å were considered. The RPD structures were aligned to the average dark state geometry using the same set of rigid residues.…”

“…These conformational changes were found to be fully reversible under regeneration of the resting state. Based on all-atom molecular dynamics simulations, Ganguly et al [45] have suggested that the release of the CTT is correlated to a transposition of His378 in the vicinity of the flavin cofactor. The authors furthermore argue that protonation of this histidine could enhance the conformational flexibility of the CTT.…”

Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor

“…Phototropins, cryptochromes, and blue light using flavin adenine dinucleotide (BLUF) proteins each bind a flavin derivative as a chromophore that undergoes covalent bond formation with a cysteine residue, electron transfer (ET), and proton-coupled electron transfer (PCET), respectively, during the photocycle. Understanding the fundamental mechanisms of naturally occurring photoreceptor proteins is important for engineering novel systems that use light as a tool to achieve noninvasive control of biological processes with high spatiotemporal resolution (5)(6)(7).…”

Coordination between Differentially Regulated Circadian Clocks Generates Rhythmic Behavior

“…In the presence of light, CRY binds to TIM and both proteins are ubiquitinated for degradation by complexes involving Ramshackle (BRWD3)/JET/Cullin4 and JET/Cullin3, respectively (Ceriani et al 1999;Koh et al 2006;Peschel et al 2009;Ozturk et al 2011Ozturk et al , 2013. Several studies suggest that light triggers the reduction of the FAD coenzyme bound within CRY, inducing a conformational change that releases the CRY carboxy-terminal tail (CTT) (Dissel et al 2004;Hoang et al 2008;Ozturk et al 2011Ozturk et al , 2008Vaidya et al 2013;Ganguly et al 2016). However, other experiments indicate that light causes CTT release through a process that does not depend on flavin reduction (Ozturk et al 2014).…”