Cryptochrome structure prediction
Most of the available crystal structures of cryptochromes from various organisms are not complete, and in many cases only the amino acid sequence is available. The available part of the currently known cryptochrome structures includes the Photolyase Homology Region (PHR), but rarely the flexible C-teminal part (CCT) - this is a serious limitation, as the CCT is known to play a significant role in the functioning of cryptochromes. Additionally, the available structures have been crystallized, and thus removed from their natural environment, which could drastically affect their three-dimensional structure.
In order to compensate for this, we employ a variety of methods to predict the true structure of the proteins. The main method used is homology modelling, which we have used to construct a model of avian cryptochrome from just the amino acid sequence.
Homology models of proteins obtained directly from web servers, can rarely be used as reliable structures for further analysis, since these models are usually not stabilized. The stability of the homology model can be established and probed through molecular dynamics simulations by monitoring the root mean square displacement (RMSD) of the protein backbone during the MD simulation.
An example of the RMSD for a homology model of European robin cryptochrome 4 can be seen above. Here the RMSD stabilises with respect to the initial structure, and with respect to the equilibrated structure, indicating that the model is stable. In the figure, the inset shows that the RMSD increase is smaller if calculated relative to the post-equilibration structure, indicative of protein stability. The small peak in the RMSD of the equilibrated structure at 0.39 ms is due to the highly flexible terminal of the PHR domain, which starts as a helix but then unfolds and refolds into an alpha-helix during the simulation.
The model of European cryptochrome 4 is based on the amino acid template of mouse cryptochrome 1, which is 91% identical to European robin cryptochrome 4. The equilibrated model can be seen below, with the FAD cofactor highlighted in red and the conserved tryptophan triad shown in purple.
This shows that a stable homology model can be obtained, however, it is still only a model of the PHR domain.
Constructing the CCT
Usually the C-terminal part of cryptochrome, the CCT, is not included in the crystal structures and cannot be added in homology models (since no template is available for this part of cryptochrome), so in order to include the CCT in our simulations, we need to construct it - and we are currently working on this problem for Arabidopsis thaliana cryptochrome 1. In this case, the missing CCT consists of 219 amino acids, and the problem of constructing this part of the protein is a matter of folding it.
Protein folding is a known problem that is very hard to solve computationally - basically because there are so many possible conformations of the protein, that it is impossible to test them all in order to find the best one (unless the system consists of very few amino acids). Thus we have generated a number of "starting structures" more or less randomly, and through extended MD simulations the structures have been converging to more optimal structures. Since we end up with many structures, we can determine the best of our CCT structures through a variety of analyses (e.g. we would expect an "optimal" structure to have minimal energy).
Structures for CCT constructed this way does not necessarily resemble the "real" structure - it may not even be close - but it will most likely result in a much more realistic model for Arabidopsis thaliana cryptochrome than one missing about 32% of its amino acids, such as the currently known crystal structure.
Using Molecular Dynamics (MD), the time evolution of biomolecular systems, such as cryptochrome proteins, can be studied, on time scales up to microseconds. The time evolution of proteins give insights into properties such as activation mechanisms, binding of ligands, stability of structure predictions and other dynamic processes that occur on the microsecond time scale.
Homology models of European robin cryptochrome
Since the crystal structure of some cryptochromes, such as those from European robin, are not known, the only way to study those proteins is to create such models computationally using an approach called homology modelling. Once a homology model is constructed, it needs to be equilibrated in an aquatic environment in order to obtain a stable structure that can be used for further investigations.
The most common way to evaluate the stability of a dynamical protein simulation, is to look at the Root Mean Square Displacement (RMSD) of the protein backbone. The RMSD is an indication of the change in structure, with respect to the initial configuration, a large RMSD means that the protein has undergone large structural changes, while a small RMSD indicates smaller changes, usually an RMSD around 3 Å is to be expected for a protein on the size of cryptochrome. An example of a small and large RMSD can be seen below.
For a homology model of European robin cryptochrome 1, the RMSD has been calculated over a 1 microsecond simulation, see below, and stabilises around 3 Å, which indicates that the model does not undergo any major rearrangements and that it has become stable after around 500 ns of simulations.
Once a stable model has been obtained, more advanced properties can be investigated.
One example of such a property could be the force required to pull FAD from the binding pocket in cryptochrome from different organisms, or finding the interaction energy between FAD and the cryptochrome protein. This can be achieved by using steered molecular dynamics simulations, where an additional force is ascribed to an atom in the FAD molecule, and by evaluating the interaction energies over the dynamic simulation where the structure is in equilibrium, in this case the last 500 ns. The interaction energy between FAD and avian cryptochrome, plant cryptochrome and insect cryptochrome can be seen in figure A below, as well as the force required to pull FAD from the cryptochromes, figure B.
These calculations show that the interaction between FAD and the protein is very similar in the three different organisms, however, the force required to pull FAD from avian cryptochrome is much larger than the force required in plant and insect cryptochrome, indicating that there are physical hinderances in avian cryptochrome that makes it difficult for FAD to enter or leave the binding pocket.
Investigation of the activation mechanism of cryptochrome 4
Once a stable homology model of cryptochrome 4 from European robin was obtained, a long molecular dynamics simulation of the structure was used to investigate the activation mechanism of the protein. The trajectory was analysed using both a principal component analysis and advanced graph theoretical methods, and these analyses revealed a network of few important amino acids in the protein structure, that facilitated the structural rearrangements resulting in the conformational changes caused by activation of the protein.