Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

doi:10.21203/rs.3.rs-3579533/v1

Download PDF

Article

Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

https://doi.org/10.21203/rs.3.rs-3579533/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Cryptochromes (CRYs) belong to the class of blue light photoreceptors and are responsible for various light-triggered functions in the circadian rhythm upon excitation of their inbuilt flavin cofactor. They are functionally distinct to the evolutionarily related photolyases, which mediate light-driven repair of UV induced DNA damages. Despite these functional differences they share a high degree of structural and sequence homology. A hallmark of cryptochromes is their flexible carboxyl-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region (PHR) are not yet fully understood and differs among different cryptochromes types. In this study we investigate the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY). Here, we focus on the highly conserved C-terminal domain harboring the FAD chromophore, to study the effect of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. By coupling a high-power LED, mounted in the source region to an ion mobility mass spectrometer, we show that D321, the putative proton acceptor of the terminal proton-coupled electron transfer event from Y373, is essential for triggering the large-scale conformational changes of helix α22 and the CTE in the lit state.

Biological sciences/Structural biology

Physical sciences/Chemistry/Physical chemistry/Biophysical chemistry

Sunlight is the primary source of energy for all living things. Depending on specific proteins and the wavelength of light, it provides the energy needed for various biological mechanisms. Photoreceptors are involved in the regulation of circadian rhythms and other light-sensitive processes. Cryptochromes and photolyases are related photoreceptors by forming the photolyase-cryptochrome superfamily (PCSF) and sharing a similar light-sensitive domain, known as the photolyase homology region (PHR).¹ Although photolyases and cryptochromes have different functions, they harbour the identical non-covalently bound cofactor flavin adenine dinucleotide (FAD) as chromophore, which absorbs blue/UV-A light in its oxidized state.²

Photolyases are responsible for repairing DNA damage caused by exposure to ultraviolet (UV) light by using light energy to revert commonly formed UV-DNA damages. These DNA repair enzymes specifically repair either cyclobutane pyrimidine dimers (CPDs) as CPD-photolyases or pyrimidine (6 − 4) pyrimidone photoproducts (6-4PPs) as (6 − 4) photolyases.³ Cryptochromes, on the other hand, are involved in the regulation of circadian rhythms and other light-dependent processes such as phototropism and the regulation of gene expression.⁴ The absorbed light energy is used to initiate a cascade of events regulating gene expression, specifically in response to changes in light, which is crucial for maintaining the balance of physiological processes that are synchronized with the day-night cycle.

Cryptochromes are found in a wide variety of organisms, including plants (pCRY), animals (aCRY), and fungi.¹ While the basic structure and function of cryptochromes is conserved across different organisms, there are some differences in their specific functions. In terms of their structural properties, they differ in their poorly conserved elongated CTE, which adopts variable lengths, potentially crucial for the respective signalling pathways. For pCRY Goett-Zink et al. showed that the CTE is associated to the PHR in its dark state conformation, although the structures of the CTE are generally assumed to be intrinsically disordered in cryptochromes.^5–7 Similar observations were made for the animal-like cryptochrome from the green algae Chlamydomonas reinhardtii, CraCRY.⁸ Besides its ability for signalling, e.g. for the regulation of the circadian rhythm, CraCRY is a true bifunctional cryptochrome by being able to repair (6-4PP) as well in its fully reduced FADH^– state.^3,8

The mechanism for electron transfer and light-triggered flavin reduction relies in most members of the PCSf on a highly conserved tryptophan triad (CraCRY: W399, W376, W322). Blue light/UV-A excitation of the FAD chromophore leads to abstraction of an electron from the nearest aromatic residue, the proximal tryptophan. In CraCRY, this initiates an electron transfer cascade within the PHR (M1-E496) via the tryptophan triad from the surface-exposed aromatic residue Y373. Here, the transfer of the electron from the phenolic moiety of Y373 to the distal tryptophan W322 is coupled to deprotonation of its hydroxyl group. The acceptor of the proton released by this proton-coupled electron transfer (PCET) event has been suggested to be the side chain of D321, with which Y373 forms a hydrogen bond.⁹ Formation of the long-lived semiquinoid FADH^• state from the oxidized state of CraCRY thereby occurs without any adduction of an external reductant. Furthermore, hydrogen/deuterium exchange mass spectrometry (HDX-MS) data indicate major conformational rearrangements at the C-terminus of the PHR and the CTE (A497-E595) as response to light.⁸ Specifically, the positioning of the α22 helix prior to the CTE region is thought to be affected, albeit exact structural evidence is still missing.³ These conformational rearrangements are implicated in the functional mechanics of these blue-light photoreceptors.

Neither cryogenic electron microscopy (cryo-EM), performed on AtCRY1 or 2, nor X-ray crystallographic studies in case of CraCRY were successful to display the structural transition of the CTE upon illumination. However, it has been shown that the CTE of various cryptochromes is involved in the circadian signalling mechanism. E.g. DmCRY undergoes a photoinduced conformational transition that allows it to interfere directly with TIM,¹⁰ while in case of AtCRY1 a light-triggered regulatory effect through a direct intermolecular interaction with COP1 upon light induced structural rearrangement was observed.^11,12 CraCRY variants with truncated CTE retain their photochemical characteristics and DNA repair activity.⁸ So far, no direct downstream signalling mechanism for CraCRY is known and the characterization of the hypothesised CTE-mediated interactions is quite challenging, because of their general flexible character.^6,7

To decipher the structural interaction and the conformational transition upon illumination, structure determining methods are needed, that are capable to handle the flexibility of the CTE, where high resolution methods like NMR or cryo-EM are limited. So far, X-ray crystallography, HDX-MS or smFRET have been employed to elucidate the conformational change of CraCRY upon blue-light irradiation, where some conformational change of the flexible CTE and/or helix α22 due to illumination has been observed.^3,8,13 Nevertheless, factors affecting the detachment of the CTE and the respective time scales of these structural transitions have not yet been comprehensively investigated.

Here, we investigated the light-induced structural transition process of full-length CraCRY by mass spectrometry. Electrospray Ionization Ion Mobility Mass Spectrometry (ESI IM-MS or IMS) is a common analytical technique, allowing the analysis of proteins under native-like conditions.¹⁴ For IM measurements the ions drift through inert gas, the resulting collisions slow them down in correlation to their three-dimensional shape. The resulting arrival time distributions (ATDs) of ions of the same mass/charge ratio can be used to calculate collision cross sections (CCSs). Under low energy drift conditions, the CCS can inform about conformational aspects of biomolecules, which can be related to their solution structure. Under higher collision energies (CE) protein unfolding can be induced. Especially controlled collision-induced unfolding (CIU) can be used to reveal a proteins gas phase stability. A change in gas phase stability can serve as a proxy to indicate a conformational change, induced for example by addition of a ligand or a change of the buffer conditions.^15–17 We modified the instrument to allow activation of CraCRY by blue light irradiation directly prior to IMS analysis. This facilitates the unambiguous assignment of the obtained CIU mobilograms which allowed us to gain a deeper insight into the factors that affect the stability of the CTE and thereby affect the function of CraCRY. Due to the relatively large time between sample ionization and ion detection, effects like proton or electron transfer, amino acid side chain reorientation or redox changes upon photoactivation, that occur on timescales of ps to ns are too fast to be picked up, but IMS is well suited to track slower, protein conformational changes that result from illumination and lit state formation. Camacho et al. demonstrated that by coupling a high performance light source to the ion source of a mass spectrometer, the evolution of the dimer and monomer signals of the UVR8 photoreceptor under UV-B illumination can be monitored using native ESI-IM-MS.¹⁸

Using this instrumental improvement, we were able to analyse CraCRY in its dark state, in its lit-state and interestingly also to follow the light-triggered transition process. Time-resolved investigation of this transition process for different mutations located close to the electron transfer chain allowed us to understand the significance of salt bridges and ionic interactions near the aspartate D321 involved in the PCET process itself and the second aspartate next to it, D323, for stabilizing the interactions between the α22 helix and the PHR as well as its effect on the CTE.

Following light-triggered conformational changes – Method validation

To illustrate, that the electrospray ionization process does not interfere with the photochemical mechanisms driving the light induced structural changes and IMS is suitable to identify those as well for determining rate constants of conformational changes upon irradiation, the well characterised photoactive yellow protein (PYP) was studied, where UV/Vis-based rate constants are available. A detailed description of the respective results can be found in the supplementary information.

Native ESI MS reveals light-triggered changes of CraCRY

CraCRY in its oxidized state appears in a wide range of charge states (CS) from 14 + to 42+, mainly distributed over two populations. The occurrence of two charge state distributions, low CS mainly between 14 + and 17 + and high CS from 34 + to 42+, suggests an overall structure of CraCRY consisting of a structured and an intrinsically disordered part, that promotes the ionization according to the chain ejection model (Fig. 2).^19,20 A denatured structure, commonly associated with higher CSs, can be ruled out as UV/Vis data show CraCRYs photoactivity and MS spectra detect the non-covalently bound FAD chromophore being part of the oxidized state. Overall, the observed charge distributions reflect CraCRY’s native state, which consists of the ordered PHR, while the CTE region is unstructured.

Formation of the FADH^• lit state causes an intensity decrease of the high charge state distribution (CSD) at 34 + to 42+, which recovers when the illumination is turned off. The decrease of the high CSD under illumination is accompanied by the rise of a third, medial CSD between 26 + and 32 + that vanishes when illumination is switched off. Together, this indicates a conformational change in an unstructured region towards a more compact state upon illumination. The light-triggered rise and disappearance of an additional conformation can also be observed in the respective driftscope plots, underlining the conclusion that the appearance of the medial CSD around 30 + reflects the semiquinoid, lit-state conformation of CraCRY (Figure S1).

As the α22 helix is expected to be the region most strongly reacting by conformational changes to light-triggering, we investigated CraCRY mutants, for which the association of this helix to the PHR predicted to be affected: The interaction of the PHR and the α22 helix was assumed to be stabilized by two salt bridges/ionic interactions between D321 and D323 on the PHR and R485 and R492 on the α22 helix respectively.^8,9 The positioning of the respective residues is shown in Fig. 1. Accordingly, mutants were chosen for which the ability to form these interactions would be affected (D321N, D321A and D323N).

Figure 2 shows mass spectra for all mutants with and without illumination. Under both conditions the mass spectra of the D321A mutant lack the high CSD, whereas the medial CSD around 30 + is present, even under dark-state conditions. Comparison with the spectra of CraCRY wildtype supports an always lit-like conformation for D321A that is independent of illumination. This result is in line with the suggested role of ionic interactions between the carboxylate group of D321 and the guanidinium groups of R485 (4.3 Å) and R492 (4.4 Å) from helix α22. These interactions are missing in the D321A mutant and apparently promote the increased mobility of the α22 helix.⁸ Interestingly, the D321N mutation was expected to similarly weaken this interaction, which would have been indicated by an increase of the medial CSD in the lit-state. However, only the generically found low CSD around 16 + could be observed, i.e. there is no shift in charge states, which would imply major conformational transition by blue light irradiation. This indicated a stabilization rather than weakening of the α22-PHR interaction and a lack of subsequent compaction of the overall conformation with the CTE. Apparently, D321 exerts a key role by acting not only as putative proton acceptor during the terminal PCET event from Y373, but also by locking in its anionic state the α22 helix to the PHR region ⁹. In contrast, the D323N mutant exerts similar MS features as the CraCRY wildtype, indicating that no significant structural alteration is associated with mutation of D323, which points to a less important role for D323 compared to D321 in terms of its stabilizing role in the α22 helix/PHR interface.

IMS analysis of light-triggered structural changes of CraCRY

To further understand the structural impacts of the different mutations we investigated their behaviour by ion mobility spectrometry. Driftscope ion mobility plots for all CraCRY mutants show several species (Figure S1), which is an indication for conformational heterogeneity. Specifically, the collision-induced gas phase unfolding of the proteins under collision energy ramping can give insight into structural changes due to illumination-triggered events and site-specific mutations.

In the following experiments the analyte ions were activated in the trap region of the mass spectrometer with subsequent drift time determination, as a function of increasing collision voltage (CV).

Figure 3 shows the CIU of the low CS 15 + for all tested mutants under non- and under illuminated conditions for collision energies ramped from 5 V to 160 V. Under low CV all CraCRY variants retain their compact structure and only undergo CIU when specific CV are reached and extended structures are getting populated, that appear at larger drift times. Dark and illuminated conditions were compared by examining the respective CIU plots of the lower charge states for all CraCRY variants (Fig. 3). These show no major differences in their unfolding behaviour, revealing no differences in intrinsic stability against unfolding.

The CraCRY wildtype shows four CIU main populations in its CIU mobilogram. The compact conformation appears at a CCS of ~ 4500 Å², which unfolds at 35 V into its first unfolded conformational ensemble of about ~ 4800 Å², followed by a second conversion at 60 V to ~ 5200 Å² and a last one at 100 V to its maximum of ~ 5700 Å². In case of the D321N mutant, the unfolding ensemble of 4800 Å² is missing and a direct conversion of the compact conformation to the second unfolding ensemble appears at a slightly larger collision voltage (55 V) compared to the wildtype. This intermediate state also differs in its smaller CCS of ~ 5100 Å² that is diminished by ~ 100 Å² compared to CraCRY wildtype. Furthermore, its final unfolded state is only weakly populated and appears very high collision voltages of about 140 V. Overall, this suggests that the D321N mutation causes a stabilization of the α22-PHR association, whose release requires higher CVs to promote unfolding of the entire PHR. This increased stability is supported over different charge states, with the CIU plots of D321N for charge state x always resembling those of charge state (x-1) of the other CraCRY mutants (Figure S2-S4). Remarkably, the D321A mutant, for which its medial CSD in the MS spectra suggests adoption of a lit-like state also in darkness, does not differ from the CraCRY wildtype in its CIU behaviour at low CS. This makes D321N the only mutant that stands out, while none of the CraCRY mutants reveals any clear differences upon illumination for their lower CS. The latter observation is not surprising as a minimal CS is usually required to reflect differences in the unfolding behaviour (see above). Therefore, we focussed next on the medial CSD (26 + to 32+), for which several additional effects can be observed. All noticeable changes occur between 5 V and 50 V and are depicted in Fig. 4. For better visualization CIU difference plots are shown additionally, which present the changes upon illumination for all species. The CIU of CraCRY wildtype starts with a compact conformation of about 7900 Å², unfolding into two almost equally populated conformations of 8500 Å² and 8700 Å² at 25 V, the smaller of which vanishes at 40 V in favour of the larger one. Unlike for the low CSs, there is a clear response visible for the medial CS (+ 30) upon illumination.

At low collision voltages we see that CraCRY compacts from 7920 to 7440 Å² upon illumination (Figs. 4A, 4B). This change becomes especially apparent in the difference plot (Fig. 4C). This illuminated state also unfolds at 25 V into two distinct unfolding species as observed for the non-illuminated conditions. These unfolded species appear at diminished CCS (8180 and 8610 Å²) compared to non-illumination (8490 and 8790 Å²). The smaller of the two unfolded conformations disappears upon increase of CE, as observed for the dark state, but interestingly already at 28 V instead of 40 V, indicating a much less stable intermediate state. The differences observed under illumination indicate a photoactivated structural change into a unique state, strongly suggesting that the light induced species at 7500 Å² reflects the lit-state conformation, whereas the population at 7900 Å² for the non-illuminated settings corresponds to the dark state.

Comparing the unfolding behaviour for the other tested variants with that of CraCRY wildtype, additional differences become apparent as seen in Fig. 4. Comparison between the CIU patterns of the D321A mutant acquired under dark and illuminated conditions show that their traces are largely identical to each other and both resemble those of CraCRY wildtype in its lit-state. As indicated before by the ESI-MS data (Fig. 2), the D321A mutant adopts a lit-like conformation independent of illumination. Consequently, the D323N mutant again exhibits major similarities to CraCRY wildtype unfolding behaviour with regard to size and stability of the present conformations, indicating no large structural effect of the mutation. Due to the lack of the medial CSD for the D321N mutant no respective CIU contour plot could be measured at CS 30+.

Delineation of MS-based rate constants for CraCRY conformational transitions

As our MS-based approach differentiates between two conformational states of CraCRY corresponding to the dark- and lit-state conformations, we can now follow its time-dependent transition. To determine the rate constants of the transition into the lit-state we illuminated the ESI tip briefly, to trigger protein excitement and structural rearrangement into the lit-state directly prior to transfer into the mass spectrometer. Ideally the illumination would occur over a timeframe much shorter than the expected reaction and back reaction, but the light energies available triggered no noticeable reaction. Therefore, the measurements were taken by illuminating the ESI tip for 3, 6 and 21 s after 9 s initial data acquisition. We then followed transition into the signalling state and back. Transitions occurring over the whole illumination phase were taken into account by a global fit, in which chromophore excitation and electron transfer are represented by rate constant k₁, the conformational change upon excitation by k₂, and the transfer back into the dark state by k₃.

Non-continuous illumination allows to track the structural rearrangement upon illumination over time, which can be followed by the irradiation-induced increase or decrease of the respective MS signals (Fig. 5A). Comparing the medial and high CSDs around 30 + and 39+, respectively, an opposing trend can be observed, indicating a direct transition between two conformational states, triggered by illumination, which is followed by the consecutive recovery into the initial state (Fig. 5B). The direct conversion between two different conformations is also trackable, comparing individual arrival time distributions over time. In Fig. 5C the same data is plotted in a non-normalized (i) and a normalized (ii) plot. The non-normalized ATDs reflect the disappearance and reappearance of the high CS 39 + in the same time frame as the opposing behaviour is seen for the medial CS 30 + in response to illumination. The normalized view indicates the concurrent compaction of the protein for the given charge states. Time courses of the other variants tested are shown in Figure S5.

Besides the light-induced compaction, which we have observed already in CIU data, comparison of the time-dependent intensity courses shows a relatively fast transition into the lit-state and a slower dark state recovery. The rate constants for k₂ and k₃ vary between 0.1 s^− 1 and 1 s^− 1, as listed in Fig. 6. The conformational changes clearly appear on different time scales than the ultrafast electron uptake of the FAD chromophore, which occurs on a sub-ps time scale followed by protonation in the µs time scale and then stays in its reduced FADH^• state for hours, depending on the respective conditions.²¹ The fast reactions related to the chromophore are reflected in our fit by the rate constant k₁ taking a value of 38 s^− 1 as initial fit parameter.²² Furthermore, our data suggest that the large-scale conformational changes upon lit-state formation initially triggered by light-driven FAD reduction, get reversed into the dark state conformation while the chromophore itself rests in the FADH^• state.

Comparison of the kinetics of the CraCRY mutants for adopting a lit-state conformation indicates that the D323N mutant exhibits a transition rate enhanced by a factor of two compared to the wildtype, which indicates some destabilisation of the α22 helix/PHR interface. This observation is in line with the crystal structure of the truncated CraCRYΔCTE, where an end-on salt bridge between D323 and R485 (OD2-NH1: 3.5 Å, OD1-NH2: 2.8 Å) contributes to the stabilization of the α22 helix/PHR interface.³ Likewise, the rate constant for dark state recovery of the D323N mutant was increased by a factor of five relative to CraCRY wildtype, indicating a favoured re-attachment of the α22 helix to the PHR. As no transition was observable for D321A and D321N, these mutants were not part of the time-dependent analysis.

ESI-IMS-MS

CCS calibration was performed under native conditions according to Ruotolo et al., using 10 µM BSA, concanavalin A and ADH.²³

MS data acquisition was performed on a Synapt G2s instrument (Waters Corpn., Wilmslow, UK) equipped with an offline nESI source and a high mass quadrupole upgrade. Au sputtered nESI tips were pulled in-house from borosilicate glass capillaries on a Flaming/Brown Micropipette Puller (P-1000; Sutter Instrument Co., Novato, CA, USA). Sample illumination was performed using a high-power diode laser (Lasertack, Fuldabrück, Germany) operating at a wavelength of 445 nm. Laser power was adjusted either to 200 or to 300 mW/cm². A home-build shutter was used to correlate the illumination according the scan time of the mass spectrometer. Capillary and cone voltages were set to 1.9 kV and 100 V, respectively. The source block temperature was set to 30°C. Directly prior to MS analysis, 10 µL of the protein solution (30 µM) was buffer exchanged into 200 mM ammonium acetate, pH 7.4 at 4°C using Amicon Ultra 0.5 Centrifugal Filter 30 kDa MWCO (Merck KGaA, Darmstadt, Germany). The rest of the settings for MS analysis were adjusted as follows: cone voltage 100 V at an offset of 100 V, 20°C source temperature. The instrument was calibrated by a conventional CsI solution. IM experiments were done using a traveling wave setup operating at a wave height of 40 V, a traveling wave velocity of 700 m/s, a nitrogen gas flow of 90 mL/min, and a drift cell pressure of 3.5 mbar. CIU experiments were performed by increasing the trap collision energy (CE) in steps of 5 V from 5 to 160 V. Data analysis of ESI-MS and IMS experiments was done using the software MassLynx V4.1, TWIMExtract²⁴, and UniDec²⁵. Theoretical CCS calculations were performed on MD structures, that were kindly provided by Yan-Wen Tan and Haiguang Liu (IMoS).

MS based rate constant determination

The rate constant determination was performed with the same settings as the ESI-IMS-MS experiment, apart from the scan time, which was set to 10 scans per s to obtain the best adjustable resolution. Sample illumination was performed for 3, 6 and 21 s for each CraCRY variant. For rate constant determination, the light triggered intensity increase and decrease of the peaks with CS 20 + to 43 + was considered by integration over the increasing MS signals divided by the sum of all used CS peak integrals. The populations assigned to dark and lit state are shown in Fig. 5A.

The corresponding data was then fitted to a kinetic model that includes three rate constants to account for the chromophore excitation and electron transfer (k₁), conformational change into the lit state (k₂) and the dark conversion process back into the conformational ground state (k₃). The model includes potential re-excitation dynamics already during the illumination period. This three-state model allows to replicate the excitation into the lit-state and the exponential decay of the signaling state into the ground state, which is observed only after a certain delay after illumination (Fig. 5B). At time points after the illumination period, no further excitation was allowed (k₁ was set to 0 s^− 1). A scaling factor was applied to compensate for small variations in the signal intensity of the different datasets. Because of the interdependence between the amplitude and the dynamic parameters in the sequential model, the parameters were fitted by alternatingly freezing the scaling factors or the kinetic constants. The fit was applied simultaneously to all datasets (3 s, 6 s, and 21 s illumination), to optimally determine the parameters that best describe the overall behavior of each CraCRY variant. The reported standard errors of the fitted parameters were determined from the Jacobian after the final fitting of the rate constants.

Sample preparation

Expression plasmids based on pET28a carrying the CraCRY mutations were generated by site-directed mutagenesis using synthetic DNA oligomers (Metabion, Germany) and were verified by sequencing (Microsynth, Germany).⁸ CraCRY wildtype and its mutants were overproduced and purified following the protocol published by Beel et al.²⁶ To get rid of any potential nucleic acid impurities binding to the protein, a heparin affinity purification step was used after the NiNTA step. For that, the protein solution was applied to the column and washed with five column volumes of a low-salt buffer containing 50 mM sodium phosphate at pH 7.8 and 20% (v/v) glycerol. The chromatography run was performed on an NGC chromatography system (Bio-Rad) using gradient elution with a high-salt buffer containing 2 M sodium chloride. For the final purification step, size-exclusion chromatography was performed in a buffer containing 50 mM NaH₂PO₄ at pH 7.8 and 100 mM NaCl.

Characterization of the reduction behavior of CraCRY mutants

All in vitro measurements were performed with a V-660 UV/Vis photometer (JASCO) at 10°C. The spectra were recorded after a desalting step with a PD-10 column for removing unbound chromophore under exclusion of light. In vitro photoreduction was performed using a blue light LED. The LED has a maximum emission wavelength of 455 nm (ILS, Intelligent LED Solutions) at a distance of 4 cm (light intensity: 5.9 mW∙cm^− 2, i.e. 224.41 µmol∙m^− 2∙s^− 1). The irradiation time was recorded in 5 min intervals.

Our MS and IMS results show a remarkable light-induced structural response of CraCRY upon illumination, implying the formation of a signalling state conformation with a change of the α22/PHR interface and the attached CTE. In this conformational rearrangement of CraCRY, which follows blue-light dependent photoreduction of FAD_ox to FADH^•, the interactions between D321 and D323 and the respective basic counterparts in the α22 helix play a key role.

Native ESI-MS spectra of CraCRY wildtype under non-illuminating conditions show mainly two different CSDs (low and high), which are indicative for the mostly folded, i.e. the PHR, and the locally disordered regions, i.e. the CTE, of CraCRY.²⁷ These two CSDs are well observed for the CraCRY mutants except D321N, which only shows the low CSD, suggesting a stabilized overall PHR structure with reduced disorder between the PHR and α22 helix. This increased stability is as well apparent in the CIU plots of CS 14 + to 17+, as the D321N CIU plots for all charge states x, resemble those of the other variants at CS x-1. This means that the same unfolding steps require the higher lab frame energies experienced by higher charged ions at the same CE. Exemplarily we see in Fig. 3 the higher CE required by D321N to undergo the first gas phase unfolding step for CS 15+, when being compared with all other CraCRY variants.

For higher charge states conformational differences become more apparent in ion mobility plots as the Coulomb interaction widens existing gaps between highly charged protein domains a bit further. Exemplarily Fig. 5 shows CIU plots for the medial CS 30 + which exhibit distinctive features in dependence of the underlying protein conformation. At low collision voltages, for which the observed CCS should most closely resemble the solution structure, CraCRY wildtype and the D323N mutant show a reduction of the CCS after illumination, while the D321A mutant matches the reduced CCS of the other variants under also in the dark state.

Looking at the entire CIU plots we observe features, which we can assign for CraCRY wildtype to the dark state (Fig. 4A) and the lit state (Fig. 4B). Qualitative comparison of the different plots now allows to distinguish those that resemble the dark state, e.g. both states for D321N and those that resemble the lit-state, i.e. for wildtype, D323N and for both states of D321A.

The time-dependent measurements allow obtaining rate constants for the conformational changes from the dark state to the lit-state and back. These time constants are clearly independent from the time scales on which the photochemistry itself at the FAD chromophore occurs (Figure S6) and differ for the analyzed CraCRY variants.^9,22

The following model for light-triggered conformational changes in this animal-like cryptochrome can explain all our MS-based findings:

Blue light excitation of the FAD chromophore in CraCRY triggers the electron transfer cascade over the conserved Trp triad (W399, W376, W322) from Y373. The PCET from Y373 causes formation of the neutral tyrosyl radical Y373° due to its deprotonation. The fate of this proton now determines the conformational differences we observed:

In CraCRY wildtype the proton is predicted to be accepted by D321, leading to the loss of the ionic interactions between D321 from the PHR and R485 and R492 from α22.⁹ A second stabilizing interaction is made by the end-on salt bridge between D323 and R485, which is not directly affected by the PCET-mediated protonation of D321. Out of these aspartic acids the latter is further away from the pivotal point connecting the α22 helix to the protein, giving the respective interaction the higher relevance in keeping the α22 helix associated to the PHR of CraCRY. Protonation of D321 disrupts its interaction with the arginines and promotes unlocking of the α22 helix and formation of the lit state conformation required for down-stream signaling.

Interestingly, the comparison of the respective mass spectra shows shifts to lower CS upon illumination, which indicates an unexpected compaction of CraCRY upon illumination, while studies on CraCRY by smFRET and small-angle X-ray scattering (SAXS) suggested a less compacted conformation for CraCRY’s lit state.²⁸ The compaction observed by us is well mirrored by the ion mobility plots, where the illuminated species show smaller CCS. As previous findings indicate CraCRY to take a larger conformation upon illumination, for example the radius of gyration (Rg) increases from 33.5 Å to 34.1 Å, one may expect this to be reflected in the mass spectra and IM heatmaps, as we observed it before for the PYP mutants, for which we performed proof of principle experiments for comparison (Figure S1-3).¹³ Those show an increase in the average charge state as well as the CCS, which both reflect a transformation to a larger conformation. The contradictory behavior of CraCRY upon illumination, i.e. compaction in the lit state vs. decompaction, when comparing our MS-based approach and smFRET or SAXS, may be a result of the different environments for assessing the light-triggered conformations. Whereas CraCRY resides in bulk aqueous solution for smFRET and SAXS, the naked CraCRY native species in IMS may undergo partial collapse of their disordered regions (CTE), which depend on the context of the folded protein moiety, here PHR. In this way, the CCS may in some cases not be directly translatable to structural data as available by X-ray diffraction or scattering. Our data suggests that the loss of the D323-R485 salt bridge and the ionic interactions made by D321 tethers the α22 helix to the PHR and allows alternative interactions with the so far disordered region of the CTE when being freed by lit-state formation. This restructuring leads to an overall smaller CCS, as opposed to the dark state, as seen in the CIU plots and reflected by the rise of the medial CSD. In any case, the CraCRY CIU plots show several CCSs, which vanish in favour of larger CCSs, as the protein unfolds, depending on the applied CV. This is not unusual in cases, where the protein structure includes several domains, which unfold stepwise. Here we observe several stepwise unfolding steps, where the second unfolding step remarkably differs for protein conformations that correspond to the lit-state.

Restructuring of the α22 helix/PHR interface and the CTE is expected to be functionally relevant for signaling by CraCRY, e.g. by enabling the recruitment of potential downstream signaling partners in the lit state. The mutation of residues responsible for stabilisation of the α22 helix/PHR interface by salt bridges/ionic interactions may affect the above-mentioned processes as follows:

The mutation D321N positions an asparagine in the proximity of the proton donor Y373. No proton transfer occurs, hindering the unfolding of the α22 helix. Given the lack of light-driven formation of the medial CSD the D321N CraCRY is apparently locked in the dark state conformation, despite photoreduction of its FAD chromophore. The ionic interactions by D321 and their disruption by light-triggered PCET from Y373 go further along the “lever arm” of the α22 helix than the second set of ionic interactions made by D323 as shown by the wildtype-like behaviour observed for the D323N mutant. Mutating D321 to alanine has the opposing effect. The D321A mutant lacks any ionic interactions stabilizing the α22 helix/PHR interface and does not participate in the PCET reaction from Y373, giving this protein a lit-like conformation also in the dark state.

To include the effect of the neighboured salt-bridge residue we investigated D323N. The ESI-MS and IMS results show a transformation of the dark state to the lit-state, albeit at a rate increased by a factor of two compared to the wildtype (wt: 0.124 ± 0.0004 s^− 1, D323N: 0.250 ± 0.0013 s^− 1). The increased rates, at which the α22 helix dissociates, can be understood in terms of the loss of the D323-R485 salt-bridge, which forms the second tether of the α22 helix to the PHR. We investigated the rate constants of the dark conversion as well, for which we see again increased rates for the D323N mutant (wt: 0.218 ± 0.0005 s^− 1, D323N: 1.075 ± 0.0042 s^− 1). This can be understood, as this asparagine only interacts via hydrogen bonds, which apparently can break and form on faster timescales than the end-on salt bridge to R485.

It is interesting to see how the single-site mutations affect the stability of the α22 helix/PHR interface, underlining the importance not only of D321 but as well of D323 with regard to the conformational change after FAD chromophore excitation. In addition, the time-resolved ESI-MS measurements allow the study of protein conformational transitions for deriving their respective rate constants. The obtained transition rates show an accelerating effect by introducing the D323N mutant, without affecting the protein stability regarding CIU. This observation is quite remarkable, especially concerning the major effect of the introduction of the D321A and D321N mutant.

In summary our data show the relevance of a few specific residues lining the α22-PHR interface for the precise timing of the rearrangements of CraCRY into the lit-state and conformational reversion into the dark state. Our findings highlight the delicate balance by which the PHR, α22 and the CTE of CraCRY are locked in place or released to allow for a partly refolded signaling state with exact time control.

Acknowledgements

We thank Yan-Wen Tan and Haiguang Liu providing CraCRY MD structures for comparisons with experimental CCS. We also thank Johann Silvestrow and Robert Hühn for their work on preliminary experiments, data processing and their helpful discussions. We gratefully acknowledge funding from the State of Hesse in the LOEWE Schwerpunkt GLUE for R.Z., LOEWE Exploration MagnetoYeast (L.-O.E.,C.J.R.) and from the research training group Complex Light Control (CLiC) at the Goethe University Frankfurt for J.S. N.M. as funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project-ID 426191805.

Author Contributions

Conceptualization: R.Z., M.S. L-O. E. and N.M.; Sample preparation: M.S., C.J.R.; investigation and methodology: R.Z., M.S., S.S. J.S, and J.G.L.; writing—original draft: R.Z. and N.M.; writing—review & editing: R.Z., S.S., M.S., J.L., J.S., C.J.R., J.B., L.-O. E. and N.M.; visualization: R.Z.; supervision and funding acquisition: N.M., L.-O.E., J.B.

Conflicts of interest

There are no conflicts to declare.

Lin, C. & Todo, T. The cryptochromes. Genome biology 6, 220; 10.1186/gb-2005-6-5-220 (2005).
Ahmad, M. & Cashmore, A. R. Seeing blue: the discovery of cryptochrome. Plant molecular biology 30, 851–861; 10.1007/BF00020798 (1996).
Franz, S. et al. Structure of the bifunctional cryptochrome aCRY from Chlamydomonas reinhardtii. Nucleic acids research 46, 8010–8022; 10.1093/nar/gky621 (2018).
Kavakli, I. H. et al. The Photolyase/Cryptochrome Family of Proteins as DNA Repair Enzymes and Transcriptional Repressors. Photochemistry and photobiology 93, 93–103; 10.1111/php.12669 (2017).
Goett-Zink, L., Toschke, A. L., Petersen, J., Mittag, M. & Kottke, T. C-Terminal Extension of a Plant Cryptochrome Dissociates from the β-Sheet of the Flavin-Binding Domain. The journal of physical chemistry letters 12, 5558–5563; 10.1021/acs.jpclett.1c00844 (2021).
Partch, C. L., Clarkson, M. W., Ozgür, S., Lee, A. L. & Sancar, A. Role of structural plasticity in signal transduction by the cryptochrome blue-light photoreceptor. Biochemistry 44, 3795–3805; 10.1021/bi047545g (2005).
Parico, G. C. G. et al. The human CRY1 tail controls circadian timing by regulating its association with CLOCK:BMAL1. Proceedings of the National Academy of Sciences of the United States of America 117, 27971–27979; 10.1073/pnas.1920653117 (2020).
Franz-Badur, S. et al. Structural changes within the bifunctional cryptochrome/photolyase CraCRY upon blue light excitation. Scientific reports 9, 9896; 10.1038/s41598-019-45885-7 (2019).
Lacombat, F. et al. Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome. Journal of the American Chemical Society 141, 13394–13409; 10.1021/jacs.9b03680 (2019).
Ceriani, M. F. et al. Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Science (New York, N.Y.) 285, 553–556; 10.1126/science.285.5427.553 (1999).
Wang, H., Ma, L. G., Li, J. M., Zhao, H. Y. & Deng, X. W. Direct interaction of Arabidopsis cryptochromes with COP1 in light control development. Science (New York, N.Y.) 294, 154–158; 10.1126/science.1063630 (2001).
Liu, B., Zuo, Z., Liu, H., Liu, X. & Lin, C. Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes & development 25, 1029–1034; 10.1101/gad.2025011 (2011).
Li, P. et al. Direct experimental observation of blue-light-induced conformational change and intermolecular interactions of cryptochrome. Communications biology 5, 1103; 10.1038/s42003-022-04054-9 (2022).
Leney, A. C. & Heck, A. J. R. Native Mass Spectrometry: What is in the Name? Journal of the American Society for Mass Spectrometry 28, 5–13; 10.1007/s13361-016-1545-3 (2017).
Vallejo, D. D. et al. A Modified Drift Tube Ion Mobility-Mass Spectrometer for Charge-Multiplexed Collision-Induced Unfolding. Analytical chemistry 91, 8137–8146; 10.1021/acs.analchem.9b00427 (2019).
Fantin, S. M. et al. Collision Induced Unfolding Classifies Ligands Bound to the Integral Membrane Translocator Protein. Analytical chemistry 91, 15469–15476; 10.1021/acs.analchem.9b03208 (2019).
Lieblein, T. et al. Structural rearrangement of amyloid-β upon inhibitor binding suppresses formation of Alzheimer's disease related oligomers. eLife 9; 10.7554/eLife.59306 (2020).
Camacho, I. S. et al. Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor. Proceedings of the National Academy of Sciences of the United States of America 116, 1116–1125; 10.1073/pnas.1813254116 (2019).
Metwally, H., Duez, Q. & Konermann, L. Chain Ejection Model for Electrospray Ionization of Unfolded Proteins: Evidence from Atomistic Simulations and Ion Mobility Spectrometry. Analytical chemistry 90, 10069–10077; 10.1021/acs.analchem.8b02926 (2018).
Beveridge, R. et al. Relating gas phase to solution conformations: Lessons from disordered proteins. Proteomics 15, 2872–2883; 10.1002/pmic.201400605 (2015).
Spexard, M., Thöing, C., Beel, B., Mittag, M. & Kottke, T. Response of the Sensory animal-like cryptochrome aCRY to blue and red light as revealed by infrared difference spectroscopy. Biochemistry 53, 1041–1050; 10.1021/bi401599z (2014).
Nohr, D. et al. Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids. Biophysical journal 111, 301–311; 10.1016/j.bpj.2016.06.009 (2016).
Ruotolo, B. T., Benesch, J. L. P., Sandercock, A. M., Hyung, S.-J. & Robinson, C. V. Ion mobility-mass spectrometry analysis of large protein complexes. Nature protocols 3, 1139–1152; 10.1038/nprot.2008.78 (2008).
Haynes, S. E. et al. Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics. Analytical chemistry 89, 5669–5672; 10.1021/acs.analchem.7b00112 (2017).
Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Analytical chemistry 87, 4370–4376; 10.1021/acs.analchem.5b00140 (2015).
Beel, B. et al. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii. The Plant cell 24, 2992–3008; 10.1105/tpc.112.098947 (2012).
Stuchfield, D. & Barran, P. Unique insights to intrinsically disordered proteins provided by ion mobility mass spectrometry. Current opinion in chemical biology 42, 177–185; 10.1016/j.cbpa.2018.01.007 (2018).
Kottke, T., Xie, A., Larsen, D. S. & Hoff, W. D. Photoreceptors Take Charge: Emerging Principles for Light Sensing. Annual review of biophysics 47, 291–313; 10.1146/annurev-biophys-070317-033047 (2018).

There is NO Competing Interest.

CraCRYnatcomSIfinal.docx
Supporting Information

Download PDF

Version 1

posted

You are reading this latest preprint version

Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

Status:

Version 1

Abstract

Figures

Introduction

Results and discussion

Following light-triggered conformational changes – Method validation

Experimental

ESI-IMS-MS

MS based rate constant determination

Sample preparation

Characterization of the reduction behavior of CraCRY mutants

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1