Conformational changes involved in signal transduction in avian cryptochromes

It is hypothesised that the putative magnetoreceptor protein cryptochrome enables night-migratory birds to navigate using the Earth’s magnetic field in a light-dependent way. Following a well-established radical pair mechanism, different downstream chemical and biochemical reactions are thought to take place depending on how the inclination of the Earth’s magnetic field influences the singlet and triplet yields of the magnetically sensitive radical pair. This is thought to lead to protein conformational changes followed by protein-protein interactions with interaction partners.
These conformational changes and interactions have been investigated as the first steps of the magnetoreception signalling process using native mass spectrometry, mass photometry, chemical crosslinking followed by mass spectrometric analysis, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The latter technique is capable of discovering interaction surfaces and conformational changes by comparing surface exposures. Recently, it was possible to detect conformational changes in certain protein regions of cryptochrome 4a of several bird species using this method, with especially prominent changes found in the phosphate-binding loop, the protrusion motif, and the C-terminal tail. Differences were seen when the experiments were performed with and without blue light. The results are detailed in chapters 3 and 4. Small changes due to an applied static magnetic field were investigated in detail in chapter 4.
Since it was recently found that avian cryptochromes can form dimers and as dimers of non-bird cryptochromes seem to play important roles in nature, chemical crosslinking was performed to narrow down the interaction surface between monomers in potentially functional dimeric cryptochrome structures, complemented by molecular dynamics simulations performed by collaborating research groups.
Two residues, C317 and C412, were found to play a role in light-induced dimerisation via disulphide bonding. To further investigate the interaction surface, electron paramagnetic resonance spectroscopy was used, more specifically double electron-electron resonance, to complement the previously acquired data with cysteine-residue-dependent distance measurements using nitroxide spin labels. The findings on dimerisation are presented in chapter 5.
Finally, by mapping conformational changes found in molecular dynamics simulations onto changes observed in a previous HDX-MS data set on the Drosophila cryptochrome, a few gaps left by the incomplete coverage of the current experimental data were filled by acquiring information on the phosphate-binding loop and the protrusion motif, two regions which have also been found to undergo light-induced structural changes for the avian cryptochromes investigated. The results of this theoretical investigation are discussed in chapter 6.
Using a range of complementary techniques, this thesis aims to elucidate protein conformational changes as the first step in signal transduction in several cryptochrome species finding similar changes in the same loop regions across multiple proteins, tackled the question whether a functional dimer might exist in the context of magnetoreception and found two important residues involved in dimerisation, and lastly used the finding that this dimerisation process is light-induced to start investigating the interaction surfaces in these dimeric structures.

Advisor: Hore, P
Benesch, J