Given its relevance as a Parkinson’s drug, and structural similarity to the endogenous ligand caffeine, we chose the A2AR antagonist istradefylline as the starting point for photoswitch development (Fig. 1). Istradefylline is known to be photosensitive and capable of photoisomerization around its central stilbene bond31, 32, 33, however, its low solubility prevented large-scale reproducible crystallization. We therefore designed and selected a series of variants (StilSwitch1-4) with the aim of improving both solubility and photoisomerization efficiency, which are key to obtaining TRSX datasets with sufficient signal to observe conformational changes. The removal of methyl groups from various points of istradefylline can increase solubility and will also alter the electronic properties of the molecules, helping to influence the photoisomerization kinetics. This type of modification was also chosen so as not to introduce bulky groups, which could interfere with the interactions of the ligand and residues of the binding pocket. For xanthine-based antagonists of the A2AR, such as istradefylline, this is a hydrogen bond with N2536.55 (superscripts refer to Ballesteros-Weinstein numbering34) and aromatic ring stacking with F168ECL2. Other investigated modifications involved replacement of the central C = C stilbene bridge with an N = N azobenzene bond (AzoSwitch1-3), as azobenzenes are known to have more efficient isomerization than stilbenes and generally shift absorption to longer wavelengths35, properties that made them prototypical photoswitches for many applications24, 29, 30.
We formulated three key properties to enable successful TRSX experiments using designed photoswitches, (Fig. 1); (I) the bound ligand structure can be obtained (a convolution of solubility, crystallization conditions and affinity); (II) a detectable effect of photoswitching on the protein and (III) efficient photoisomerization in the protein binding pocket (which can significantly differ from solution). Based on these properties, candidates from a pool of commercially available and custom synthesized istradefylline derivatives were evaluated through cryo-crystallography and a series of biophysical assays, which provided a basis for deciding whether to continue a photoswitch candidate through the characterization pipeline.
Crystallographic characterization of istradefylline derivatives
Prior to biophysical characterization of the istradefylline derivatives, crystal structures were obtained to ensure modifications to the ligand did not prevent binding in the orthosteric pocket. This also enabled solubility for crystallization to be assessed. AzoSwitch1 and AzoSwitch3 were ruled out from further characterization, due to their low solubility causing precipitation of the compounds. Crystals soaked with the remaining ligands diffracted to resolutions of 1.95–2.35 Å (Extended Data Table 1), allowing the binding interactions of the ligands to be clearly identified and demonstrating that modification of the istradefylline scaffold did not prevent ligand binding (Fig. 2). The binding pose was similar for all photoswitches and was typical of xanthine-based antagonists of the A2AR37. Additionally, some derivatives formed water mediated hydrogen bonds with the carbonyl group of S672.65. All ligands also interacted with E169ECL2, either through π-hydrogen bonds or water-mediated hydrogen bonds. E169ECL2 forms part of a lid over the A2AR binding pocket38 together with T2566.58 and H264ECL3. Previous studies have demonstrated that stabilization of the ionic interaction between E169ECL2 and H264ECL3 through ligand interactions can produce longer residence times39. Therefore, the removal of this stabilization after isomerization is likely to cause a reduction in affinity through the modification of dissociation kinetics. The inhibition constant of cis istradefylline has previously been measured and was observed to be lower than that of the trans state33, supporting the idea that the cis states of the istradefylline derivatives are likely to have lower affinities.
Assessing the effect of photoswitching on the protein
A photoswitch designed for use in TRSX must detectably change the protein-ligand interaction to induce conformational changes upon illumination. As it is well known that GPCRs are stabilized by the binding of high-affinity ligands40, we used changes in thermal stability to assess the magnitude of changes in protein-ligand interactions. A simple method available in most labs for studying these changes is differential scanning fluorimetry, which takes advantage of fluorescent dyes to monitor the temperature-induced unfolding of proteins41. The unfolding of GPCRs in these assays is typically monitored using 7-diethylamino-3-(4’-Maleimidylphenyl)-4-methylcoumarin (CPM), which interacts with cysteines in the unfolded protein and fluoresces at 470 nm41. Unfortunately, the absorption of AzoSwitch2 interferes with this wavelength and prevented the collection of thermal shift data. For all stilbene-based photoswitches, we generally observed a progression of the melting curve back towards the stability of the apo state upon illumination: both in terms of melting temperature, and the rate of onset of the melting transition (Fig. 3A). The reduction in thermal shift of illuminated relative to dark samples demonstrates that illumination causes a decrease in the thermal stabilization of the A2AR by the photoswitch, which can be interpreted to correlate approximately with photoswitch affinity.
Whilst monitoring thermal shifts is a simple method to assess protein-ligand interactions, it is static and cannot assess temporal changes. Nevertheless, the impact of the derivatization of istradefylline on the change in thermal shift can be observed clearly for StilSwitch2-4, all of which have modifications reducing the size of the benzoyl ring relative to istradefylline. The smaller the size of the benzoyl ring, the slower the rate of thermal shift decrease after illumination: indicating that this moiety must be instrumental in determining the lower affinity of the cis state through changing the interactions of the ligand with the binding pocket.
Isomerization efficiency characterization
TRSX requires a high level of photoactivation to visualize signals in isomorphous difference maps. Photoswitch isomerization efficiencies are typically measured in the absence of protein. However, the influence of protein binding on isomerization efficiency also needs to be taken into consideration. To determine relative photoisomerization efficiencies, the absorption spectra were measured using UV/Vis spectroscopy before and after one minute of constant illumination in the presence and absence of protein (Fig. 3B). A larger decrease in trans peak absorbance following illumination would be characteristic of a higher efficiency of photoconversion. Protein binding lowered the efficiency for all isomerizing ligands, supporting previous conclusions that protein binding can reduce the quantum efficiency of photoisomerization42. Interestingly, only small changes were observed for AzoSwitch2, despite azobenzenes typically isomerizing with a higher efficiency than stilbenes35. This observation was later shown to be due to a short half-life of the cis state, resulting in thermal relaxation back to the trans state before measurement (Extended Data Fig. 1), revealing the importance of kinetic ligand characterization already at this point.
Three derivatives had higher isomerization efficiencies than istradefylline in the protein bound state: StilSwitch2, StilSwitch3 and StilSwitch4. Comparing the size of the benzoyl moiety, which was the group determined to have the strongest influence on cis state affinity, with the effect protein binding has on photoisomerization efficiency (Extended Data Fig. 2), it is clear that reducing the size of the photoswitch improves photoisomerization when bound to the protein. Whilst increasing the probability of cis state formation was a desired property, when comparing the results of UV/Vis spectroscopy to differential scanning fluorimetry, it is clear that this increase in cis state formation came at the expense of eliciting large changes in protein interactions following isomerization due to the reduction in clashes formed with the binding pocket. Together, these results demonstrate that isomerization efficiency alone should not be used to select photoswitches for use in TRSX. Instead, photoswitches should have a balance of high-yielding isomerization in the protein binding pocket, and large differences in protein-ligand interactions following isomerization. StilSwitch3 and StilSwitch4 balance these effects, given that their isomerization efficiency is not dramatically reduced by protein binding (compare ultrafast data on StilSwich3), photoswitching occurs with good yields and the total decrease in thermal shift following illumination was the largest for these photoswitches.
Kinetic characterization of photoswitch candidates
A time-resolved spectroscopic characterization of the photoswitch derivatives was carried out to probe isomerization dynamics in more detail. Transient absorption spectroscopy shows that at around 10 ps, the stilbene-based photoswitches exhibit an additional state in aqueous solution (Fig. 4E & F), which was not observed in acetonitrile (Extended Data Fig. 4). The intensity of the new state scales in the order StilSwitch1 < StilSwitch3 < StilSwitch4 < StilSwitch2, also visible in the transient (Fig. 4E). Here, we see a rising intensity around 10 ps associated with this state. Time needed for populations of the additional state follows the same order as the intensity. StilSwitch3 shows the highest remaining signal, while StilSwitch1 shows the lowest isomerization yield (Fig. 4E & F). This result resembles the steady state spectra when illuminating at 340 nm. Remarkably, StilSwitch2 and StilSwitch4 (highest novel state concentration) show red-shifted steady state fluorescence spectra (Extended Data Fig. 3F), indicating another radiating species. Based on this analysis, an excited state branching is likely. One part of the excited population undergoes isomerization, while the second part most likely forms a conformationally relaxed intramolecular charge transfer (CRICT) state. This state is well known for push-pull-stilbenes and decays via fluorescence43, 44, 45, 46, 47.
Looking into the kinetics of the photoreaction of StilSwitch3 (Fig. 4A), we observe a broad excited state absorption, extending out to 100 ps. The stimulated emission (405–450 nm) overlaps with the CRICT state while the ground state bleach (< 400 nm) signal is constant at 2 ns, indicating successful isomerization. The lifetime density analysis yields four different lifetime distributions at 1 ps, 4 ps, 40 ps and > 2 ns (Fig. 4B). While the first density accounts for excited state absorption (ESA) and the decay of stimulated emission (SE) and consequently partial ground state bleach (GSB) recovery, the second, negative distribution accounts for formation of the CRICT state. Within the third component, the ESA and the CRICT state decay, while the last distribution indicates product formation. Within the constraining protein, the decay of StilSwitch3 is slowed (Fig. 4C & D) and the CRICT state formation is no longer visible. To obtain the key populations and states involved in the photoreaction, we performed a global target analysis with a sequential model (Fig. 4G). The best fits were achieved with four different lifetimes. The corresponding evolution associated difference spectra (Extended Data Fig. 5A) show the involved states and have very common spectral features, only the intensities vary. This supports the idea of a strongly decreased photoreaction (Extended Data Fig. 5B) combined with a strongly non-exponential behavior. Remarkably, the CRICT state formation is inhibited by the protein environment, which prevents this inactivation pathway. On the other hand, the isomerization rate is also slowed down and reduced by the protein environment. Since the photoreaction proceeds beyond the measurement time window, we cannot observe complete isomerization here, however, the steady state data (Fig. 3B) demonstrate a trans to cis photoreaction. In combination with the steady state data, it can be concluded that the process has lower rates and lower quantum yields within the protein binding pocket, as is often the case for photoswitches under confined conditions42, 48, 49, 50, 51. Overall StilSwitch3 showed the highest photoconversion in steady state, while having only a very small formation of the non-productive CRICT state, suggesting it as an ideal candidate to test its photoreaction when bound to the A2AR.
Assessing behavior of photoswitches in crystallo with serial crystallography
After balancing characteristics of the designed istradefylline derivatives (Fig. 5), we selected StilSwitches2-4 as promising candidates for serial synchrotron crystallographic experiments. Following protocols, we developed for the study of light-activated rhodopsins25, 52, 53, 54, we collected serial crystallographic datasets under constant illumination at the Swiss Light Source. In the case of StilSwitch4, crystals did not yield sufficient diffraction patterns under these steady-state conditions. Also, in the case of StilSwitch2 and StilSwitch3, light exposure reduced the rate at which diffraction patterns could be collected. Nevertheless, crystals diffracted well enough for the collection of light-activated datasets (Extended Data Table 2) with resolutions of 2.8 Å and 3.25 Å, respectively.
Difference electron density maps and structural refinements against the data show clear trans-cis isomerization around the stilbene C = C double bond (Fig. 6A, B). Even though both ligands only differ by a single methyl group on the tilting benzoyl side chain, the photoinduced change had somewhat unexpectedly different effects on the ligand binding pocket. Of particular interest in this regard is the hydrogen bond network formed by the triad of E169ECL2, T2566.58, and H264ECL3 and the hydrophobic pocket above Y2717.36 that have been suggested as key sites during multistep ligand dissociation in the A2AR based on mutagenesis and molecular dynamic simulations38.
In the case of both StilSwitch2 and StilSwitch3 the resulting cis-state clashes with Y2717.36 and causes it to move away from the binding pocket (Fig. 6A, B). In StilSwitch2 these changes shift the position of the hydrogen-bonded lid on the binding pocket (E169ECL2, H264ECL3, T2566.58) without opening it. Key features of the cis state-bound binding pocket observed with StilSwitch2 are also present for StilSwitch3. However, the clash with Y2717.36 at the base of ECL3 is more pronounced, likely because the extra methyl group increases the size of the benzoyl moiety. These changes partially break the hydrogen bond between H264ECL3 and E169ECL2 opening the suggested exit pathway between ECL2 and ECL3 (Fig. 6B, D-F)38. These structural results explain the larger change in thermal shift upon illumination of StilSwitch3 and suggests that the extra methyl group on the benzoyl ring changes dissociation kinetics. This difference potentially allowed us to trap two different intermediates of the dissociation pathway, further emphasized by a shift in the sodium ion, know to modulate A2AR activity and ligand binding55, 56, only observed in StilSwitch3 (Fig. 6C). Cholesterol is another allosteric regulator of A2AR activity with state-dependent binding sites57, 58, 59. Interestingly, illumination of StilSwitch3 causes a loss of cholesterol from the antagonist bound A2AR binding sites, whereas, for StilSwitch2 we observe less negative difference density over the cholesterol molecules (Extended Data Fig. 6). The loss of cholesterol from these sites indicates a movement of the A2AR away from its inactive, antagonist-bound state. Based on these results we concluded that ligand unbinding is more efficient when using StilSwitch3 and selected this variant for further analysis by time-resolved crystallography.
Time-resolved synchrotron crystallography of ligand dissociation
For the collection of time-resolved serial crystallographic data we used the recently established Kilohertz serial crystallography setup60 at MaxIV. Due to the increased flux at this next-generation synchrotron source, we collected data with a temporal resolution of 2 ms over a window ranging up to 70 ms after the reaction was initiated with a laser pulse (Extended Data Fig. 7). To follow light-induced changes over time, we calculated a series of difference electron density maps (Fo(light)-Fo(dark)) each representing 2 ms in time and subjected them to Pearson correlation analysis26, 27 (Fig. 7A). The analysis revealed two major states, one ranging from approximately 2–40 ms and a second state contributing from 50–70 ms at the end of the measurement. Based on difference electron density maps calculated from the corresponding temporal ranges (Fig. 7B, C), the first state contains isomerized ligand in the cis conformation confirming our previous results in a time-resolved setup. In the case of the second state, near continuous negative density covers the position of the ligand, demonstrating that the transient cis-compound has left the binding pocket. The H264ECL3-E169ECL2 salt bridge has been broken and additional difference density can be found throughout the receptor (Fig. 7B, C). This result demonstrates that the designed photoswitch is a viable trigger to induce larger conformational changes throughout the receptor in accordance with the dynamical nature of GPCRs.