Changes in active site histidine hydrogen bonding trigger cryptochrome activation Abir Gangulya, Craig C. Manahanb, Deniz Topc, Estella F. Yeeb, Changfan Linb, Michael W. Youngc, Walter Thiela, and Brian R. Craneb,1 a Max-Planck-Institut für Kohlenforschung, 45470 Mülheim an der Ruhr, Germany; bDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; and cLaboratory of Genetics, The Rockefeller University, New York, NY 10065

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). dCRY engagement of TIM and JET depends on conformational changes at the dCRY C-terminal tail (CTT). In the dCRY resting structure, the CTT forms a helix that binds into the flavin cofactor pocket (6–8). In the presence of light, proteolytic sensitivity of the CTT increases (7–10) and the flavin becomes more exposed to redox agents (7, 10). Removal of

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the CTT destabilizes dCRY but also promotes dCRY-mediated TIM degradation via JET (11). The mechanism by which light causes conformational change in the dCRY CTT is not well understood, and there is uncertainty regarding which electronic states of FAD respond to light and promote CTT displacement. dCRY purifies from insect cells with an oxidized flavin, which then undergoes photoreduction to the anionic semiquinone (ASQ) (10, 12, 13). As with other CRYs and PLs, a conserved triad of Trp residues reductively quenches the FAD excited singlet state to produce the ASQ (14). Both photo- and chemical reduction to the ASQ have been shown to affect the conformation of the CTT and promote interaction with a peptide derived from TIM (10). However, other studies suggest that light excitation of the oxidized flavin cofactor alone is sufficient to produce the conformational changes necessary for target recognition (9, 15). Most of the CTT forms a 10-residue helix (527–536) that contains three consecutive aromatic residues: the Phe534–PheP535– Trp536 (FFW) motif (7, 8, 10, 16). The FFW residues bind in the flavin pocket, but are separated from the cofactor by a conserved His residue (His378). In some PLs, the analogous His has been shown to undergo changes in protonation state during the catalytic cycle (17, 18). Herein we investigate the relationships among flavin redox state, His378 protonation state, and CTT conformation by using a combination of computational and experimental methods. The results indicate that His378 protonation promoted by flavin photoreduction disrupt CTT interactions with the protein core. Results dCRY Conformational Behavior in Different Redox and Protonation States. To study dCRY at various stages of photoactivation, we

considered different redox states of the flavin and protonation Significance There are few detailed mechanistic models that describe how light-sensing proteins convert photochemistry into conformational signals. Combined computational and experimental investigations reveal how photoreduction of the Drosophila cryptochrome (dCRY) flavin induces protonation of a neighboring conserved His residue. Altered His hydrogen bonding then leads to conformational change in a key regulatory element of the protein. These data provide further evidence for involvement of a flavin anionic semiquinone in the dCRY signaling state and allow for the design of variants that can be used to separate dCRY biological functions or as tools for optogenetics. Author contributions: A.G., C.C.M., D.T., E.F.Y., C.L., W.T., and B.R.C. designed research; A.G., C.C.M., D.T., E.F.Y., C.L., and B.R.C. performed research; A.G., C.C.M., D.T., E.F.Y., C.L., M.W.Y., W.T., and B.R.C. analyzed data; and A.G. and B.R.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence may be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1606610113/-/DCSupplemental.

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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 pKa. 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.

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Edited by Donald G. Truhlar, University of Minnesota, Minneapolis, MN, and approved July 11, 2016 (received for review April 26, 2016)

Fig. 1. The dCRY resting state. (A) The starting conformation of dCRY derived from the crystal structure [Protein Data Bank (PDB) ID code 4GU5; flavin, green bonds; CTT, red; C-terminal lid, yellow; phosphate binding loop, purple; protrusion motif, orange; C-terminal base loop, blue]. (B–E) For oxidized flavin (FAD), the preferred conformation of His378 (B and C) and its RMSD with respect to the crystal structure (D and E) position over the course of three trajectories (red, blue, and green). B and D correspond to His378 protonation at position N3; C and E correspond to His378 protonation at position N1. When His378 is N3 protonated (B), it forms a stable hydrogen bond between His N1 and FAD OS2 and remains very close to the crystal conformation (orange sticks). When His378 is N1 protonated (C), the His N1FAD OS2 interaction is disrupted; His378 rotates out of its crystal position and interacts with the FAD adenine N1 through His N3.

states of His378 (Fig. 1A). To identify the resting state of the dCRY active site, we performed MD simulations with oxidized flavin and His378 protonated either at the N3 position (His-N3P) or protonated at the N1 position (His-N1P; Fig. 1 B and C). The RMSDs of His-N3P and His-N1P from the crystal structure configuration of His378 are quite different along their respective MD trajectories (Fig. 1 D and E). His-N3P retains its position from the crystal structure, with a stable hydrogen bond between unprotonated N1 and the ribose hydroxyl OS2 of FAD (Fig. 1B). The close correspondence of His-N3P to the crystal structure is reflected in near zero values of the corresponding residue RMSDs. By contrast, in the trajectories with His-N1P, the His378 N1 -FAD OS2 interaction is disrupted because of N1 protonation; instead His-N1P moves out of its crystal position and N3 accepts a relatively stable hydrogen bond from FAD N1 of the adenine moiety (Fig. 1C). Correspondingly, the His-N1P RMSD assumes values of 2–3 Å in each of the independent trajectories (Fig. 1E). The computed RMSDs suggest that His378 is predominantly N3 protonated in the resting state of dCRY. To elucidate the structural and dynamical changes triggered by reduction of the flavin, we then performed MD simulations with the flavin in the anionic semiquinone state (FAD—), and His378 in either the neutral His-N1P, neutral His-N3P or the doubly protonated imidazolium state (His-DP). In the FAD—:His-N3P case, increased motion in His-N3P was observed in two of the three independent trajectories (Fig. S1). In these trajectories, flavin reduction weakens the FAD— (OS2)-His-N3P interaction to an extent that allows His-N3P to leave the active site pocket and drift toward the FFW motif. Interestingly, in one trajectory, the CTT releases such that the FFW motif moves far away from FAD— and Trp536 flips outward away from the active site (Fig. S1C and Movie S1). It is important to note here that, although the simulations are sufficiently long enough to study the equilibrium properties of the system in a defined stable state, the CTT release is a large conformational change; given the wellknown sampling-related limitations associated with classical MD simulations, such processes are not likely to occur in every independent trajectory. Nonetheless, surmounting the barrier for substantial rearrangement of the CTT in response to flavin reduction and His378 protonation is clearly possible in these classical MD simulations. 10074 | www.pnas.org/cgi/doi/10.1073/pnas.1606610113

In the FAD—:His-DP simulations, the FAD— (OS2)-His-DP(N1) interaction breaks and His-DP moves freely in the active site because of the absence of suitable hydrogen-bonding partners (Fig. 2 and Fig. S2). In these trajectories, the His-DP displaces away from FAD— and moves close to the FFW motif, and in one of the trajectories we observe a similar CTT release (Fig. S2C and Movie S2). Analogous FAD:His-DP simulations with oxidized flavin show similar behavior (Fig. S2), with CTT release in one of the three independent trajectories (Fig. 2). We also examined the effects of flavin reduction on dCRY when the active-site histidine is in its His-N1P form. In these FAD—:His-N1P simulations, no large conformational changes are observed. The CTT remains in its closed conformation and HisN1P maintains the interaction with FAD adenine N1 through unprotonated His N3, which is thus held away from the FFW motif (Movie S3). The inability of His-N1P to show CTT release on flavin reduction supports the hypothesis that the resting state of His378 is predominantly His-N3P. Principal Component Analyses of dCRY Dynamics. A principal component analysis (PCA) of the FFW motif (residues 534–536) reveals the concerted motions of the CTT. Visual inspection indicates that the first eigenvector (first PC) corresponds to the CTT release. Projection of the various MD trajectories onto the first eigenvector shows that CTT release is observed for only two protonation states of His378, namely His-N3P and His-DP (Fig. 2 and Fig. S3). For His-N3P, it occurs only in presence of FAD—, whereas for His-DP, it is observed for both redox states of the flavin. CTT release is highly correlated with the position of His378 in the MD trajectories (Fig. 3). Plotting projections of each trajectory on the first PC against the position of His378 relative to the flavin and the FFW motif indicates that in the FAD:His-N1P scenario, His-N1P remains very close to FAD and does not interact with the FFW motif (Fig. 3A). In this protonation state of His378, reduction of flavin has little effect on the motions of His378 and the FFW motif (Fig. 3B). In the FAD:His-N3P case (Fig. 3C), the His378 remains localized close to the crystal configuration. There is no significant motion in the FFW motif. However, when the flavin is reduced to the ASQ (Fig. 3D), His378 motion increases (as depicted by the wider distribution in Fig. 3D), with His-N3P at times moving close to the FFW motif. This shift toward the FFW motif is more prominent when His378 is doubly protonated (HisDP) for both redox states of the flavin (Fig. 3 E and F). In all of these cases, there is substantial motion of the CTT, thereby suggesting that displacement of His378 toward the FFW motif facilitates CTT release. To explore correlated motions in dCRY, we computed dynamical cross-correlation maps (DCCMs) for the various MD

Fig. 2. CTT release in dCRY. The first PCA mode that corresponds to the CTT opening is plotted along the trajectories from the MD simulations with (A) FAD reduced and His378 N3 protonated, (B) FAD neutral and His378 protonated at both N1 and N3, and (C) FAD reduced and His378 protonated at both N1 and N3. In each panel, the sudden transitions of the respective PCA modes from negative or neutral to positive values indicate the CTT release. The red, blue, and green traces correspond to three independent trajectories. (D) Initial displacement of the CTT (red) relative to the flavin (green) and His378 (orange) observed in simulations.

Ganguly et al.

Fig. 3. CTT release correlates with the position of His378. The first PCA mode that corresponds to the CTT opening is plotted against the relative position of His378 in the active site from the MD simulations with (A) FAD oxidized and His378 N1 protonated, (B) FAD reduced and His378 N1 protonated, (C) FAD oxidized and His378 N3 protonated, (D) FAD reduced and His378 N3 protonated, (E) FAD oxidized and His378 protonated at both N1 and N3, and (F) FAD reduced and His378 protonated at both N1 and N3. The red, blue, and green colors correspond to three independent trajectories. The His378 shift on the x axis is defined as the difference between the separation of His378 center of mass to FAD center of mass and the separation of His378 center of mass to FFW center of mass. Snapshots of the active site from the various simulations are shown next to respective panels and indicated by dotted arrows. Crystal structure position of His378 is shown in orange sticks.

MD simulations, we carried out independent replica exchange simulations for the three most relevant stages of dCRY photoactivation as suggested by the previous MD trajectories, namely the “resting state” (FAD:His-N3P), the “flavin reduced state” (FAD—:His-N3P), and the “His protonated state” (FAD—:His-DP). Fig. 5 A–C depicts the distributions of RMSD of the CTT tail with respect to its closed conformation in the crystal structure, as obtained from the three independent replica exchange simulations. In the resting state (Fig. 5A), the RMSD distribution of the CTT shows a sharp peak close to 1.5 Å, suggesting that the CTT remains for the most part in the closed conformation of the crystal structure. In the flavin reduced state (Fig. 5B) and the His protonated state (Fig. 5C), the RMSD distribution is significantly broader, thereby illustrating the increased mobility of the CTT in these two states. As in the case of the MD trajectories, independent PCA analyses of the FFW motif were performed on each replica exchange ensemble. In each case, the first PCA mode corresponds to the CTT release. Fig. 5 D–F illustrates the correlation of the CTT release with the position of His378 for each of the three states. As was observed in the MD trajectories, in the resting state (Fig. 5D), the His378 remains close to the crystal conformation and there is little CTT movement. In the flavin-reduced state (Fig. 5E), the His378 still remains close to its crystal position but some CTT release is observed. In the His protonated state, His378 shifts from its initial position, which results in occasional CTT release (Fig. 5F). The frequent CTT release observed in the REMD simulations allows us to estimate the free energy differences between the open and closed states in each case from the relative population of the two states in the respective REMD ensemble (Table S1). If the CTT displacement threshold for separating closed and open conformations is chosen to be the first peak in the CTT RMSD Ganguly et al.

Flavin Reduction Promotes His378 Protonation. To investigate whether flavin reduction thermodynamically favors a particular His378 protonation state, we performed Poisson–Boltzmann/linear response approximation (PB/LRA) calculations to determine the pKa shifts of His378 in the protein environment compared with aqueous solution. These shifts were computed at the N1 and N3 positions for both redox states of the flavin. PB/LRA calculations have been previously shown to successfully reproduce the experimentally determined pKa shifts of a variety of protein residues and RNA nucleobases (19). In the present work, they were done for two different protein dielectric constants, eprotein = 2 (Table S2) and eprotein = 4 (Table S3); the results from each were qualitatively

BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Replica-Exchange Simulations. To substantiate the findings from the

distribution of FAD:His-N3P (Fig. 5A) ΔG(closed → open) is calculated to be 0.81 kcal/mol for the FAD:HisN3P case, −0.08 kcal/mol for the FAD—:HisN3P case, and −0.37 kcal/mol for the FAD—:His-DP case. Thus, although FAD:HisN3P favors the closed state, FAD—:HisN3P slightly favors the open state and FAD—:His-DP substantially favors the open state.

CHEMISTRY

trajectories (Fig. 4 and Fig. S4). A DCCM constructed from a trajectory with reduced flavin (FAD—), the doubly protonated His378 (His-DP), and that produces CTT release, illustrates the regions of dCRY that are highly correlated to the CTT (Fig. 4 and Fig. S4). Not surprisingly, several of these regions are proximate to the CTT. The qualitative picture of the DCCMs is similar for all MD trajectories (Fig. S4).

Fig. 4. Correlated motions in dCRY as reported by the DCCMs. Different regions are colored according to their correlation with the CTT (yellow), with the degree of correlation shown by color shading from least correlated (dark blue) to most correlated (red). Stars show trypsin cleavage sites that either become more (yellow) or less (cyan) accessible in light.

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pH Dependence of dCRY Photoreduction. If the FAD—:His-DP state

Fig. 5. REMD simulations also reveal CTT mobility. (Left) Distribution of the CTT RMSD with respect to its crystal conformation with (A) oxidized flavin and His378 protonated at N3, (B) reduced flavin and His378 protonated at N3, and (C) reduced flavin and His378 protonated at both N1 and N3. For oxidized flavin and N3 protonated His378, the CTT is stable in its closed conformation as suggested by a sharp RMSD peak at ∼1.5 Å (A). When either the flavin is reduced or the His378 is doubly protonated, the CTT is highly flexible as suggested by the broad RMSD distribution ranging from ∼1 to 7 Å (B and C). (Right) Correlation plots between the CTT opening and the relative position of His378 in the active site from REMD simulations. The 2D probability density (high-red; low-blue) of CTT opening and His378 shift is illustrated for the simulations with (D) oxidized flavin and His378 protonated at N3, (E) reduced flavin and His378 protonated at N3, and (F) reduced flavin and His378 protonated at both N1 and N3. The His378 shift on the x axis is defined as in Fig. 3.

similar. Beginning with FAD:His-DP, it is more favorable to deprotonate N1 than N3, which is consistent with the resting state involving a proton on N3 and a hydrogen bond between unprotonated N1 and the ribose hydroxyl of FAD. This preference for single protonation on N3 (N3P) over N1 (N1P) holds true for both the oxidized and reduced form of the flavin. When the flavin is reduced to the ASQ, the doubly protonated His378 (His-DP) is stabilized relative to either of the singly protonated states. The transition from N1P to DP (i.e., with protonation of N3) produces the largest average pKa shift (4.0 units; Table S2); however, in the resting state, which as discussed earlier, is hypothesized to have His378 protonated at N3, the relevant pKa shift is from N3P to DP (i.e., protonation of N1). In this latter case, a substantial average shift of 1.1 pKa units of His378 accompanies formation of FAD— (Table S2). Free energy perturbation (FEP) calculations were also used to estimate the His378 pKa shift when the flavin is reduced (SI Materials and Methods and Table S4). As with the PB/LRA calculations, both N1 and N3 upshift their pKa values in the presence of FAD—; however, the results from the FEP simulations indicate a reverse trend, with N1 showing greater upshifts than N3. It is encouraging that both the PB/LRA and FEP calculations indicate that His378 will become protonated on flavin reduction; we consider the PB/LRA calculations more reliable owing to the known convergence issues of the FEP methods for systems undergoing large conformational changes, as is the case of CTT displacement in dCRY (SI Materials and Methods). 10076 | www.pnas.org/cgi/doi/10.1073/pnas.1606610113

is thermodynamically favorable, the rate constants for its lightdriven formation could show a pH dependence. It is well known that a series of Trp residues (“the Trp triad”) reduce the dCRY flavin exited state (14). Although the rate constants for the initial electron transfer reactions between the flavin-excited state and Trp residues are large, steady-state accumulation of the ASQ depends on several other factors that include the quantum yields of flavin excited state formation, the illumination conditions, recombination reactions, and reductive quenching by external reductants. Furthermore, the observed photoreduction rate constant (kobs) depends not only on the flavin reduction rate constant (kf) but also on the reoxidation rate constant (kr), where kobs = kf + kr. Under our conditions of illumination, the dCRY flavin reduces to the anionic semiquinone with a rate constant (kobs) that is largely first order. Reoxidation (kr), measured separately, is also comparatively slow (i.e., kobs ∼ kf). Under these conditions, the rate of flavin reduction progressively increases as pH drops from 9.5 (0.1 s−1) to 6.5 (0.5 s−1; Fig. 6A). Thus, increasing proton availability accelerates flavin reduction, even though the reduced flavin itself does not become protonated. Rather, stabilization of the increased negative charge, as reflected by a modest increase in net reduction rate, is achieved by protonation in the vicinity of the flavin, i.e., on His378 (we note that a very small amount of the neutral semiquinone forms at lower pH and is likely responsible for a small secondary phase in the kinetics; Fig. S5). When His378 is changed to Asn, photoreduction is slower than for WT at low pH and is no longer pH dependent. CTT release also accompanies ASQ formation, and this conformational change may hence influence the value kobs, i.e., destabilization of the CTT by the His378Asn substitution could also contribute to an altered photoreduction rate. dCRY in its dark-adapted and light-adapted states have characteristic tryptic digestion patterns that reflect the conformation of the CTT (10). Examining these patterns for lightand dark-adapted dCRY as a function of light exposure indeed reveals that the CTT has reduced stability in the Asn378 variant (Fig. 6B). MD simulations run on His378Asn dCRY show that in all trajectories, Asn378 does not hydrogen bond to other residues in the active center and is itself very mobile, despite maintaining a potential hydrogen bond acceptor (the amide carbonyl group) in a similar position as unprotonated N1 of His (Fig. S6). For the oxidized flavin the His378Asn CTT remains rigid, close to the crystal conformation in only two of three trajectories; in the other, the CTT changes conformation, but to a lesser extent than seen with the WT protein on His protonation or flavin reduction (Fig. S6). When the flavin is reduced, similar changes in the CTT occur in two of three trajectories (Fig. S6). Thus, the His378Asn substitution alone destabilizes the CTT, in both the resting and photoreduced forms of dCRY. We examined the behavior of a His378Arg substitution that places a stable positive charge in the vicinity of the flavin. As might be expected, His378Arg dCRY shows little pH sensitivity to photoreduction and releases the CTT more readily in the light (Fig. 6 A and B). However, the structural effect of Arg substitution is uncertain, given that the large side chain will not be well accommodated by the crystal structure conformation. Effects of His378 Substitution in Cellular Assays. The biological activities of His378 dCRY variants were tested by cotransfecting Drosophila S2 cells with epitope-tagged dCRY, TIM, and JET and then examining the dark and light-stabilities of both TIM and dCRY (Fig. 6C). As suggested by the proteolytic assays, the His378Asn mutant is less stable in both the light and the dark. Consistent with this variant being partially activated owing to destabilization of the CTT, more TIM is degraded in both the light and the dark, than with WT dCRY (Fig. 6C). Thus, the inability of the Asn378 variant to protonate is convoluted with its increased activation and decreased stability in cell culture. Ganguly et al.

Discussion In investigating the conformational activation mechanism of dCRY, we focused on a conserved His residue that juxtaposes both the flavin and the CTT. The corresponding residue is known to undergo changes in protonation state for closely related photolyase enzymes during their catalytic cycle (17, 18). dCRY clock function is not known to involve DNA repair, and hence, this potentially ionizable residue may be conserved for another reason, perhaps linked to the formation of the ASQ by dCRY. MD simulations of dCRY suggested that in the resting state His378 should be protonated at the N3 position. In these simulations, His-N3P remained localized close to the crystal structure conformation, where it forms a stable hydrogen bond between unprotonated His378 N1 and an FAD ribose hydroxyl group. In contrast, when His378 was in its alternative tautomeric state, namely protonated at N1, the residue rotated out of its crystal conformation and moved further away from the CTT to interact with the adenine moiety of FAD. The His-N3P state was found to be sensitive to ASQ formation. Flavin reduction weakened the hydrogen bond between the ribose hydroxyl of FAD— and unprotonated His378 N1, thereby allowing His378 to leave the active site pocket and drift toward the CTT. We hypothesize that movement of His378 facilitates CTT release based on the fact that conformational changes at Ganguly et al.

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Similar effects are not seen with the His378Gln variant, which appears to behave much like WT for both dCRY and TIM degradation (Fig. 6C). Interestingly, the His378Arg and His378Lys substitutions give a striking phenotype: in both cases, TIM degradation is normal, but there is no light-induced dCRY degradation (Fig. 6C). Given that Arg and Lys have quite different structures from His and from each other, the impact of such substitutions on CTT conformational stability and conformation may be complex. Nonetheless, the behavior of His378 variants in cellular assays underscores the sensitivity of dCRY to the charge and hydrogen-bonding patterns of residue 378.

CHEMISTRY

Fig. 6. Photoreduction, CTT release, and cellular activity of dCRY His378 variants. (A) Photoreduction rate constants for light-induced formation of the ASQ are pH dependent for WT dCRY (blue diamonds), but not for the His378Asn (red circles) or His378Arg (orange triangles) variants. Under these conditions, reoxidation recovery rates are negligible. Only rate constants for the dominant phase are shown. Simulation of a simple model that assumes a single deprotonation event fits the WT data with a pKa = ∼7.7. (B) Extent of light-induced CTT release as measured by proteolytic sensitivity for the WT, His378Asn, and His378Arg variants after increasing times of light exposure. Error bars reflect the SD for n = 3. (C ) Effect of His378 substitutions on dCRY stability and dCRY-mediated TIM degradation in insect cell culture. Quantification of relative protein levels from Western blot analysis of TIM (Upper) and dCRY (Lower) expressed with JET in S2 cells under light or dark conditions.

the CTT were observed only for the His-N3P: FAD— case and never for the His-N3P:FAD, His-N1P:FAD, or the His-N1P: FAD— cases. However, the CTT release was also observed when His378 was doubly protonated for both redox states of FAD. In these cases, His-DP has no suitable H-bonding partners in the active site and is often pushed toward the CTT. Thus, the inability of the active center to simultaneously provide hydrogen bond acceptors to both N1 and N3 counters the favorable electrostatic interaction of the positively charged His side chain beside the negatively charged flavin. The position of His378, which is thus related to the protonation state of the residue, was found to correlate highly with motion of the CTT, which packs against His378. These observations suggest that His protonation to the imidazolium is sufficient to restructure the CTT. It follows that flavin photoreduction drives His protonation by increasing the negative charge of the active center. pKa calculations were performed to identify the effect of ASQ formation on His378 protonation states. We observed a positive pKa shift at both N1 and N3 positions of His378 when FAD is reduced, which is consistent with the hypothesis that ASQ formation is accompanied by His378 protonation. Interestingly, the pKa shifts were found to be higher at position N3 than N1 with the PB/LRA treatment; this could imply that there are two potential channels of CTT release. Channel 1 represents a scenario in which His378 is predominantly N3 protonated and ASQ formation is sufficient to affect the CTT release, with or without additional His protonation. Regardless, the pKa calculations and photoreduction experiments indicate that His378 will ionize on flavin reduction. Thus, the doubly protonated state is the most destabilizing for the CTT conformation, as also indicated by the REMD free energy calculations. Channel 2 represents a small fraction in which His378 is initially N1 protonated. In this case, ASQ formation alone is not sufficient for CTT release, but it triggers His378 protonation, which in turn facilitates the CTT release. Cross-correlation analyses of the MD simulations revealed regions in dCRY that have highly correlated motions with those of the CTT. These regions are in good agreement to those that have been determined experimentally to have increased proteolytic sensitivity on flavin reduction (10). Small-angle X-ray scattering data indicate that the oxidized and reduced forms of dCRY do not differ greatly in their overall structure (10), which is also consistent with the simulations in that large scale conformational changes in dCRY, apart from the CTT region, were not observed. The results from the MD simulations were verified further with REMD simulations. The REMD simulations, performed only for the His-N3P:FAD, His-N3P:FAD—, and His-DP:FAD— cases, indicated that the CTT was much more flexible in the latter two cases, with His-DP:FAD— especially favoring an open conformation. The position of His378 in the active site was found to be correlated with the CTT release, as was also observed in the MD simulations. The difference in pH-dependent photoreduction for WT compared with His378Asn or His378Arg dCRY indicates that His378 protonates in response to ASQ formation. Despite the inability to further ionize, the Asn378 variant shows light-dependent activity in cell culture that exceeds that of WT, as well as some TIM degradation activity in the dark. The MD simulations indicate that this activity arises because the CTT is destabilized by a lack of hydrogen bonding by Asn378. This change in stability potentiates His378Asn toward activation in cell assays, where it shows enhanced degradation of TIM in the light and the dark. In contrast, the His378Gln substitution has properties closer to WT, including its capability to light-induce TIM degradation. The behavior of His378Gln, which cannot undergo protonation, supports the ability of flavin reduction to destabilize the His-N3P conformation without further protonation in MD simulations. Given that substitutions at His378 produce proteins that are still active in cell culture, one may wonder why His378 has been

so highly conserved. The answer likely involves the many roles of dCRY in the cell and properties such as dark state stability that affect those functions. His378 may be especially well suited to switch between stable “on” and “off” conformations. Furthermore, the His378Arg/Lys variants have very different behavior from WT with respect to dCRY degradation in the light. Thus, the dCRY conformational changes that are required to trigger these two processes may be mutually exclusive. Notably, a different E3 ubiquitin ligase (Brwd3) (5) is required for dCRY degradation as opposed to TIM degradation (JETLAG), and it follows that different aspects of dCRY restructuring may be required for interactions with each. His378Arg appears to release the CTT more readily than WT, but features of the CTT conformational change needed for dCRY activation toward TIM or self-degradation may not be well distinguished by the proteolytic sensitivity assays. Alternatively, the 378 residue itself may participate directly in the interactions that promote dCRY self-degradation in light. Importantly, these new variants provide useful tools for delineating the roles of light-induced TIM turnover from dCRY turnover in clock resetting. Moreover, plant CRY proteins are being used extensively in optogenetic applications to control molecular interactions in cells with light (20). dCRY may be similarly useful, particularly if variants provide for altered responses with partners. Recently, plant cryptochrome (AtCRY2) has also been shown to undergo light-induced changes in protonation states of active1. Chaves I, et al. (2011) The cryptochromes: Blue light photoreceptors in plants and animals. Annu Rev Plant Biol 62:335–364. 2. Conrad KS, Manahan CC, Crane BR (2014) Photochemistry of flavoprotein light sensors. Nat Chem Biol 10(10):801–809. 3. Crane BR, Young MW (2014) Interactive features of proteins composing eukaryotic circadian clocks. Annu Rev Biochem 83:191–219. 4. Koh K, Zheng X, Sehgal A (2006) JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312(5781):1809–1812. 5. Ozturk N, VanVickle-Chavez SJ, Akileswaran L, Van Gelder RN, Sancar A (2013) Ramshackle (Brwd3) promotes light-induced ubiquitylation of Drosophila Cryptochrome by DDB1-CUL4-ROC1 E3 ligase complex. Proc Natl Acad Sci USA 110(13): 4980–4985. 6. Zoltowski BD, et al. (2011) Structure of full-length Drosophila cryptochrome. Nature 480(7377):396–399. 7. Levy C, et al. (2013) Updated structure of Drosophila cryptochrome. Nature 495(7441):E3–E4. 8. Czarna A, et al. (2013) Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function. Cell 153(6):1394–1405. 9. Ozturk N, Selby CP, Annayev Y, Zhong D, Sancar A (2011) Reaction mechanism of Drosophila cryptochrome. Proc Natl Acad Sci USA 108(2):516–521. 10. Vaidya AT, et al. (2013) Flavin reduction activates Drosophila cryptochrome. Proc Natl Acad Sci USA 110(51):20455–20460. 11. Busza A, Emery-Le M, Rosbash M, Emery P (2004) Roles of the two Drosophila CRYPTOCHROME structural domains in circadian photoreception. Science 304(5676): 1503–1506. 12. Berndt A, et al. (2007) A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome. J Biol Chem 282(17):13011–13021. 13. Oztürk N, Song SH, Selby CP, Sancar A (2008) Animal type 1 cryptochromes. Analysis of the redox state of the flavin cofactor by site-directed mutagenesis. J Biol Chem 283(6):3256–3263. 14. Kao Y-T, et al. (2008) Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase. J Am Chem Soc 130(24):7695–7701. 15. Ozturk N, Selby CP, Zhong D, Sancar A (2014) Mechanism of photosignaling by Drosophila cryptochrome: Role of the redox status of the flavin chromophore. J Biol Chem 289(8):4634–4642. 16. Zoltowski BD, Gardner KH (2011) Tripping the light fantastic: Blue-light photoreceptors as examples of environmentally modulated protein-protein interactions. Biochemistry 50(1):4–16. 17. Hitomi K, et al. (2001) Role of two histidines in the (6-4) photolyase reaction. J Biol Chem 276(13):10103–10109. 18. Schleicher E, et al. (2007) Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase. J Biol Chem 282(7): 4738–4747. 19. Archontis G, Simonson T (2005) Proton binding to proteins: A free-energy component analysis using a dielectric continuum model. Biophys J 88(6):3888–3904. 20. Shcherbakova DM, Shemetov AA, Kaberniuk AA, Verkhusha VV (2015) Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools. Annu Rev Biochem 84(1):519–550.

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site moieties that are coupled to conformational activation. In this case, an Asp residue adjacent to the flavin N5 position delivers a proton to the photoreduced cofactor to obtain the neutral semiquinone state (NSQ) characteristic of the plant CRY signaling state (21, 22). The ASQ of dCRY requires no such protonation, but flavin reduction instead directs the proton to a neighboring His residue to alter interactions of the dCRY-specific CTT motif. In conclusion, MD simulations and experiments both reveal that flavin photoreduction indeed promotes conformational change at the dCRY C terminus through a mechanism that involves altering the hydrogen bonding pattern of His378. These changes are most efficiently propagated when His378 undergoes coupled protonation to the imidazolium. Materials and Methods SI Materials and Methods details the materials and methods followed in this article. The section contains description of classical MD simulations, principal component analysis, pKa calculations under the Poisson–Boltzmann/linear response approximation, replica-exchange MD, dCRY expression and purification, spectroscopic and kinetic analyses, proteolytic sensitivity assays, and cell culture stability assays of dCRY and TIM. ACKNOWLEDGMENTS. A.G. thanks Matthias Heyden for advice concerning PCA. This work was supported by NIH Grants GM079679 (to B.R.C.) and GM054339 (to M.W.Y.), and Training Grant T32-GM008500 (to C.C.M.).

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