Following the recent crystallization of a Meta-F intermediate of Agp2-PAiRFP2 with a 2.16 Å res-olution, new insight was gained in the structural changes that lead to Pr from Pfr state. This new structure opened the opportunity for computational simulations to better understand the role of
this important intermediate in the overall photocycle. We investigated the role of protonation of residues in the chromophore binding pocket on the secondary structure transition in the tongue and further assessed their pKa values and protonation population via constant pH calculations.
4.2.1 Secondary structure transition in the tongue
In bathy phytochromes, Meta-F, following Lumi-F state, is a more advanced state towards Pr than Pfr, the chromophore has already isomerized, but the tongue still retains anα-helix conformation.
We run several long accelerated MD simulations in order to observe the secondary transition in the tongue taking place. We then examined the factors influencing the transition, from protonation states to chromophore conformation.
Preparation
The Pfr model was built from the wild type (WT) crystal structure (PDB entry 6G1Y) with gaps filled and sidechains of those residues minimized with SwissModel software. BV chromophore and crystal water were inserted after alignment with the crystal structure.
The Meta-F model was built using the Agp2-PAiRFP2 Meta-F state crystal structure (PDB entry 6G20) as template and back mutated to the wild type sequence using SwissModel software. Chain A and B were modelled separately starting from the two different chains in the crystal structure, since there are some differences in the two chains in the crystal structure. The Meta-F head-to-head dimer was built aligning the two chains previously obtained with the two in the Pfr-WT crystal structure and then BV chromophore and crystal water incorporated. In this way we obtained a structural model of Meta-F state with the canonical head-to-head dimeric conformation resembling the Pfr, since the dimer found in the Agp2-PAiRFP2 Meta-F crystal structure is probably a crystallographic artifact.
To investigate the coupling between protonation states and secondary structure transitions, we tested different models where BV propionic side chain C and His278 adopt different protonations:
Table 4.2.1: Agp2 states with different protonation models tested. ”Prot” represents a singly proto-nated and ”Deprot” a completely deprotoproto-nated Prop-C, while ”ε” and ”P” indicate if His278 is proto-nated on theεposition or doubly protonated. Correspondingly we assigned a short name tag to each model.
Simulation set up
All simulations were done with AMBER ff14SB, with BV parameters from ref.[82], and run on Am-ber18 software. Each model was solvated in a water box with 12 Å edge distance from the protein and counter ions added till reached charge neutrality with Amber tool19’s leap program. Simu-lations were run on a single GPU with a 2 fs time step with periodic boundary conditions, PME for electrostatic interactions, a cutoff of 12 Å for the van der Waals interaction and hydrogens con-strained with the SHAKE algorithm. Each model was minimized for 40 ps, heated from 0 to 100 K (NVT) in 60 ps and then from 100 to 300 K (NPT) in 80 ps and finally pre-equilibrated at 300 K (NPT) for 120ps. Using as starting structure the one resulting from pre-equilibration, 1000 ns GaMD (dual boost) simulations were performed for each model at 300 K (NVT), with σ0=10 kcal/mol. We used this rather high acceleration value in order to greatly enhance sampling and be able to observe theα →βtongue transition, a process that happens on very long time scale, but without denaturating the remains of the protein.
Results
Secondary structure transition is observed in the tongue in two meta-F models, MF1 and MF4. For the MF1 model, transition happens in two runs out of five, but only once in the MF4 model. The transition involves only one of the two chains, without preference for one of the two, but simulta-neous transition of both chains is not observed in any trajectory. Theβ-confomation accounts for about 20% of the total trajectory in MF1 run 1, 30% in MF1 run 5 and 70% MF4 run 5.
The model MF4 with Prop-C deprotonated, which should have then transferred the proton to His278, is regarded as the conformation most likely leading to Pfr, but there is no evidence of favouring the secondary transition more than the MF1 model where BV Prop-C are still protonated.
425
Figure 4.2.1: Agp2 Meta-F, model MF1, run 1. Secondary structure change observed in tongue of chain A, left, from α-helix toβ-sheet, highlighted in yellow. Secondary structure plot for chain A, up right, and chain B, down right, show that only chain A undergoes and keeps the transition for about 200 ns.
425
Figure 4.2.2: Agp2 Meta-F, model MF1, run 5. Secondary structure change observed in tongue of chain B, left, from α-helix toβ-sheet, highlighted in yellow. Secondary structure plot for chain A, up right, and chain B, down right, show that this time only chain B undergoes the transition and keeps it on and off for about 200 ns in total.
Figure 4.2.3: Agp2 Meta-F, model MF4, run 5. Secondary structure change observed in tongue of chain A, left, from α-helix toβ-sheet, highlighted in yellow. Secondary structure plot for chain A, up right, and chain B, down right, show that chain A undergoes the transition as a double β-sheet and keeps it for almost 800 ns.
The other two Meta-F models, MF2 and MF3, also counting five runs each, didn’t exhibit any significant change in the tongue, which was expected. This hints that their protonation pattern is unsuitable for promoting secondary structural transition in the tongue.
Of the two Pfr models tested, none showed secondary transition in the tongue, as predictable due to the fact that Pfr conformation and side chains orientation naturally favours a tongue’sα-helix conformation.
Table 4.2.2: Agp2 models, number of runs and their results. Numbers in last column refers to the number of runs where theα→βis observed, x means is not observed in any of the run.
State Model # runs α→β
Meta-F MF1 5 2
MF2 5 0
MF3 5 0
MF4 5 1
Pfr PF1 5 0
PF2 5 0
An important factor to consider is the chromophore orientation. Since during the GaMD sim-ulation no restrain was applied to BV and as a high acceleration was chosen, the cromophore has an
increased mobility, especially ring D, which frequently switch fromαtoβfacial andviceversa, and even rotate and briefly assume a Pfr-like ZZE conformation. During such conformations the proba-bility of secondary structure transition in the tongue is decreased and the probaproba-bility for transition is considerably higher when the chromophore adopts a ZZZ conformation. An example of ring D dihedral angle is plotted in fig.4.2.4 as function of simulation time and refers to the simulation in fig.4.2.1. Chain A experience a secondary transition that spans the timescale including 400 to 700 ns, which correspond to ZZZ conformation with dihedral values of around±175°. This correlation is observed also in the other simulations where there is a secondary structure transition, but it can’t be ascribed solely to the ring D configuration.
Figure 4.2.4: Meta-F with two different BV conformations, Pfr-like (a) andα-facial Pr-like (b), adopted during the same simulation.
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Figure 4.2.5: Dihedral angle for ring D during the simulation of Agp2 Meta-F model MF1, run 1. In solid color is every 10th point, faint line is every point.
Conclusions
We have seen that a secondary structure transition in the tongue is achievable in some Meta-F mod-els, as a result of the increased conformational space explored during the GaMD simulations. This indicates that the equilibrium between theα-helix andβ-sheet conformation in the tongue is liable, but a complete transition of the whole tongue is not observed, which would require even longer timescale and specific conditions.
Protonation states of Prop-C and His278 play a role in the probability of transition, as no significant transition is observed in 2 out of 4 Meta-F models, but also an impact is played by the orientation of the chromophore that has to adopt a Pr like ZZZ conformation. This suggests that what influences the secondary structure transition cannot be attributed to a single factor, but to a combination of different elements, such as side chains protonation and conformation.
No transition is observed in any Pfr model, were BV still has a ZZE conformation, which confirms its incompatibility for a secondary structure in the tongue.
This behaviour suggests that the more likely driving force to the secondary transition in the tongue is a electrostatic change in the pocket, following the rotation of ring D and the deprotonation of pro-pionic side chain C and consequent protonation of, most likely, His278. This electrostaic change is sufficient to perturb the delicate equilibrium in the tongue and cause the transitionα→ β. The ex-act conditions for this transition are still to be explored, as no complete transition is observed in our simulations and a complete reorganization of the tongue would probably require a specific confor-mation kept for and extended time. These observations are in good agreement with the conclusions
drawn by A. Krasovet al.in their 2020 paper[103], where they argue that initial BV conformational switch initiates the destabilization of the tongue and the following deprotonation of propC com-pletes the tongue’s secondary structure transition. In the paper they rule out a direct proton transfer in the tongue region as the trigger of the secondary structural change, but instead argue that is the electrostatic change in the pocket that perturbs the stability of the tongue and induces a transition.
4.2.2 Constant pH calculations
Protonation states of the chromophore and surrounding residues are of primary relevance for the photoconversion of phytochromes. Unfortunately protons are not resolved by X-ray crystallogra-phy and our knowledge of protonation states is based only on spectroscopic studies, which are lim-ited to the chromophore binding site[104]. A method that can help in the assignment of protona-tion states based on pKa values is constant pH calculaprotona-tion in which pKa values of selected residues are evaluated and then titrated at fixed intervals during the simulation. This method has been ap-plied in several Agp2 models to try to uncover a possible proton pathway from the chromophore to the surrounding residues.
Following a recent work on Agp2 by Krasovet al.[103], where they investigated the consequences of substituting Arg211, Tyr165, and His278 to the intramolecular proton transfer in the chromophore binding pocket and the Pfr-to-Pr photoconversion with various spectroscopic techniques, such as RR, IR, UV-vis and ultrafast pump−probe experiments, we applied the cpH method to these mu-tants to have a direct experimental comparison.
Model preparation
The Pfr model was built from its crystal structure (PDB entry 6G1Y) with gaps filled with the Swiss-Model software. BV chromophore and crystal water were inserted after alignment with the crystal structure.
The Meta-F model was built using the Agp2-PAiRFP2 Meta-F state crystal structure (PDB entry 6G20) as template and back mutated to the wild type (WT) sequence using the SwissModel soft-ware. Chain A and B were modelled separately starting from the two different chains in the crystal structure. The head-to-head dimer was built aligning the two chains previously obtained with the two in the Pfr-WT crystal structure and then BV chromophore and crystal water incorporated.
Point mutations of Agp2 Pfr and Meta-F monomer variants were done by residue substitution in Pymol, using as starting structure Chain A as previously modelled for the dimer.
The Meta-Fβstarting model was taken from a snapshot of a GaMD run of the Meta-F structure in which the tongue of chain A has undergone a secondary structural change to aβ-sheet on one of the two strands (see previous section).
The Pr-like model was built using the Agp2-WT sequence and the Pr state of the Agp1-PCM (PDB entry 5HSQ3) as template. Again the head-to-head dimer was built aligning the two chains with
the two in the Pfr-WT crystal structure and then BV chromophore and crystal water incorporated.
Preparation for the constant ph calculation was done by assigning some key residues as titrat-able: BV propionic side chain C, His 248, His 278, Tyr and Cys, as described in ref [105]. Asp and Glu residues were not included following a test run, in which, at pH 7, they were exclusively depro-tonated. In order to save the computational time required to their titration, they were not assigned as titratable, as they would not give any significant contribution to the protonation pattern.
Computational details
The various Agp2 models were solvated in a water box and counter ions added to neutralize the total charge with the Amber tool leap program. The AMBER ff14SB was used with the BV parameters as described in ref [82]. All simulations were performed on GPUs with a 2 fs time step under periodic boundary conditions with the particle-mesh-Ewald method for electrostatic interactions, a cutoff of 12 Å for the van der Waals interaction and hydrogens constrained with the SHAKE algorithm.
The setup was minimized for 40 ps, heated from 0 to 100 K (NVT) in 60 ps and then from 100 to 300 K (NPT) in 80 ps and finally equilibrated at 300 K (NPT) for 120ps. From the last pre-equilibrated structure three 200 ns constant pH conventional MD was performed with AMBER parmemd.cuda software. The pH was set at pH=7 and the Monte Carlo protonation state change attempts every 100 fs.
Results
In the Pfr, Prop-C has relatively high pKa values, around 9 (see table 4.2.3), and consequently is almost never deprotonated, as reflect by the corresponding population percentage (table 4.2.3).
His248 has a pKa value around 4.5 and is mostly singly protonated, either inεorδposition, de-pending on the orientation of the ring that can easily rotate during the simulation. His278 has pKa values of about 2 units higher than His248 and has a significant population percentage of doubly protonated, but is not the most populated state, which still is the singly protonated. Water molecules surrounding Prop-C (see fig. 4.2.6) influence pKa values and could be a possible bridging proton transfer. W1 (fig.4.2.6) interacts mainly with His248 and His278, but since is highly mobile can easily leave the pocket and being replaced by another water molecule later in the simulation. W2 and W3 are stabilized by Arg211 and Ser253 and less mobile, since more deeply embedded in the pocket, but can still eventually exchange with bulk water, as observed after about 100 ns.
PKa values for the Meta-F model are in the expected range, with Prop-C values lower than in the Pfr model at around 7, so more likely to lose the proton, clearly reflected by a higher deprotonated population percentage (table 4.2.3). His248 has lower pKa values than His278, which is doubly protonated for most of the time, with a population up to 85%, while His248 is preferentially pro-tonated inδposition. PKa and population values are probably influenced also by the presence of
water in the pocket that interact directly with Prop-C, as shown in fig.4.2.6. During the simulation these water molecules move, with W1 moving between His248 and His278 and eventually leaving the pocket after 5-30 ns, depending of the run, and sporadically being replaced by a water molecule from the bulk. Water W2 and W3 are stabilized by the proximity of Arg211 and Ser260, nonetheless W2 was observed leaving the pocket after 50 ns in one simulation. The presence of W2 and W3 in the pocket, prevents direct proton transfer with the residues in the immediate vicinity and help sta-bilize the Prop-C via hydrogen bond, while W1 interfere with the transfer with the histidines and its absence promote deprotonation of Prop-C and protonation of, mainly, His278. Water molecules, on the other hand, can also act as bridging proton acceptor-donor, but this effect cannot be seen since they are not titratable during the simulation.
Meta-Fβ, despite technically being in a more advanced conformation towards Pr, presents higher pKa and a much lower deprotonated population for Prop-C than the Meta-F. This is due to the strong rearrangement of side chains and chromophore during the GaMD simulation, which strongly influences electrostatic in the pocket. This suggests that, despite the relevant change in the tongue, this is not a model that well approximates an advanced Meta-F state and is unsuitable for accurate cpH calculations.
The pKa values for the Pr model are similar to the Meta-F ones, but does not confirm the trend of a lower pKa and mostly deprotonated Prop-C. The secondary structure reflect what is expected to be a Pr state, but the side chains orientation, especially the ones inside the chromophore binding pocket, are a simple adaptation of the Agp2 sequence to the Agp1 Pr template and evidently not a good enough fit to properly asses their protonation states.
Figure 4.2.6: Water molecules in the pocket interacting with Prop-C.
An interesting conformation that emerged during the Meta-F simulation involves an H-bond between the two propionic side chains, as shown if fig.4.2.7. This state is held for few nanosecond and it’s a seldom adopted conformation, representing about a 2% of all sampled conformations.
The internal H-bond also rearranges the interactions with the two arginines in the pocket, Arg242, which usually interacts with one bond with each of the two oxygens in prop-B, now forms two H-bonds with one of the two, because the other is stretched out to form one with Prop-C’s hydrogen.
Arg211 is shifted away and instead of establishing an H-bond with the terminal guanidino group, forms a weak H-bond with the side amino group. The pKa for Prop-C of chain B associated with this conformation is 7.5, which is about the average value resulting after 200 ns, but does not contribute significantly to the final result since it’s a rarely adopted conformation.
This conformation was not observed during the Pfr simulation, confirming that it doesn’t play a significant role in the hydrogen bond network inside the chromophore binding site.
Figure 4.2.7: H-bond between the two propionic side chains and their interactions with two surround-ing Arg211 and Arg242 in the Agp2 Meta-F state.
Table 4.2.3: Mean pKa and population values after three (two for Meta-Fβ and Pr) separate 200 ns cMD cpH for the Agp2 dimer.
Chain A Pfr
We performed constant pH calculations on the Agp2 monomer wild type and some mutations in Pfr and Meta-F states. The values obtained from a monomer do not differ substantially from the dimer ones, justifying the decision to use the monomer in order to extend the sampling to several mutations that would have required much more computational time. We avoided calculations on the Meta-Fβand Pr state, since the quality of those models demonstrated to be insufficient for the calculation of accurate pKa values.
The choice of the mutants is based on the 2020 paper by Krasovet al.[103], so we can have a direct comparison with experimental results.
Table 4.2.4: Mean pKa values and population distribution after three independent 100 ns cMD cpH for the Agp2 monomer.
WT Pfr pKa Pfr Pop Meta-F pKa Meta-F Pop
The pKa values in the WT monomer are similar to the dimer and confirm the trend of a lower pKa from Pfr to Meta-F for Prop-C. An increase in pKa values of both His248 and His278 is ob-served in the same transition. The passage from a value below 7 to one above it, makes doubly protonated the most likely state for His278 in Meta-F.
The variant HIP278 refers to the wild type, but now His278 is kept doubly protonated and is not
The variant HIP278 refers to the wild type, but now His278 is kept doubly protonated and is not