4.3.1 Two dimers
Eukaryotic phytochromes are postulated as being dimers[4]. They are almost invariantly crystal-ized as head-to-head dimer, which is thought to be functional for the activation of the two facing output modules[88,99]. However there are some exceptions, most notably the head-to-tail Cph1 dimer[106], which is regarded as physiological active. Some other unconventional dimer confor-mations could arise from mutations applied to the protein or the unique conditions of high concen-tration and packing found in crystals. Then it is important to assign critically a dimer as physiolog-ically functional or as a crystallographic artifact. On this line of thought we assessed the stability in solution of a XccBph head-to-tail dimer as found in a recent crystal structure by Otero’s lab versus a more conventional head-to-head model of the same system.
Preparation
For the G454E mutant full length head-to-tail model we used the crystal structure as reported (PDB entry: 7L59). The structure include the PAS9 output module, bended about 90 with respect to the PHY domain, breaking theα-helix back spine. This unusual OM conformation, together with the mutation at the tongue and crystallographic conditions, give a antiparallel head-to-tail dimer with interface at the tongue region.
Figure 4.3.1: XccBph G454E dimer (PDB entry: 7L59). The PAS9 output module (in red) is bent circa 90, which impairs a head-to-head dimer formation.
In order to compare dimer stability, we build a head-to-head dimer resembling the full length WT dimer in Pr state, using as template the 6PL0 for dimerization, but keeping the sequence with the G454E mutation and the Pfr secondary structure, including the tongues inα-helix, from the 7L59 structure. Practically, we have filled the few gaps and optimized side chains in the G454E crys-tal structure with SwissModel, restored the PAS9 OM to the canonical position and then aligned the two monomer with the two in the Pr structure, obtaining the final, more traditional, head-to-head dimer.
Figure 4.3.2: XccBph G454E head-to-head dimer model, based on the quaternary structure of the Pr crystal (PDB entry: 6PL0). In this model the backbone helical spine is preserved and the two OM interlock with each other in an anticlockwise fashion (top view).
Simulation setup
The two XccBph models were solvated in a water box of 12 Å from the edges of the protein 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 hydro-gens 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 pre-equilibrated at 300 K (NPT) for 120ps. From the last pre-equilibrated structure a 500 ns GaMD (dual boost) simulations were performed for each model at 300 K (NVT), withσ0=5 kcal/mol. Electrostatic Energy Calculations were done with APBS[107], with 545 grid points in the{x,y,z}dimensions and a resolution of 0.3 Å.
Results
Both dimers maintain their conformation throughout the 500 ns of the simulation and no significant secondary structural transition is observed. The head-to-head model, thanks to the interlocking OMs, is quite stiff, with very little movement of the two PHY domains, as prevoiusly observed in the DrBph photosensory dimer. Fluctuations are propagated along the helical spine, but no drastic domain movement is observed, as visible in figure 4.3.3.
Figure 4.3.3: XccBph head-to-head dimer model. Panel A is the initial structure, and panel B is the final structure after 500 ns GaMD. The dimer interface is conserved with some minor rearrangement of the PAS (in green) and the OM PAS9 (in yellow) domains. The surface is colored based on the electro-static values obtained with APBS, ranging from -5, in red, to +5, in blue, units are KbT/ec.
The head-to-tail asymmetric dimer have higher domains fluctuations and an overall higher mo-bility, as highlighted by a higher RMSD compared to the head-to-head dimer (see table 4.3.1). To account for differences in the monomers interactions while in the dimer, we counted the number of formed salt bridges and H-bonds. The head-to-head dimer has a higher number of both interac-tions, but only by a small margin (table 4.3.1).
Configurational energy is a bit more negative in the head-to-head dimer, indicating a slightly more favourable dimer conformation in solvent, but binding energy is more positive than the crystal dimer, which suggest a bigger electrostatic repulsion. The reason why binding energy is positive is due to the high charge borne by each monomer. Upon dimer formation the two monomer, which would tend to electrostatically repel in solution, will continue to give a positive (repulsive) coulomb
interaction, compensated by the presence of other stabilizing interactions such as salt bridges and
Table 4.3.1: Average values of RMSD, in Å. Number of formed salt bridges and H-bonds, configura-tional and binding energy, in Kcal/mol.
Conclusions
Both dimers are stable in solution for the entirety of the simulation, without any dimer interface break or denaturation. The head-to-head dimer appears as marginally more stable, more rigid and with more interface interactions. The head-to-tail dimer assigned in the crystal structure is also en-ergetically stable, although with a slightly higher flexibility reflected by the increased mean RMSD value of about 1 Å. This dimer, despite differing considerably to the physiologically accepted head-to-head dimer, like the one we built, present all the characteristic tertiary structures and chromophore conformation of a phytochrome in Pfr state. A series of experimental evidences, such as UV-VIS, SLS-SEC measurement and electrophoresis [108], substantiate the validity of such head-to-tail construct. Therefore a new mechanism for the transition from Pr to Pfr was proposed, now called
”joint-inversion model”, in which, 1) BV’s ring D isomerize upon light absorption, 2) reorganization of side chains inside and in proximity of the chromophore-binding pocket,3) tongue transition, 4) PHY domains reorientation, 5) dimer dissociation, 6) PAS9 reorientation, and finally 7) antiparal-lel dimer formation. Similar mechanisms is postulated for the back reaction, with formation of the initial parallel dimer.