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Journal Article

Changes in active site histidine hydrogen bonding trigger cryptochrome activation

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Ganguly,  Abir
Research Department Thiel, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Thiel,  Walter
Research Department Thiel, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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pnas.201606610SI.pdf
(Supplementary material), 2MB

pnas.1606610113.sm01.mpg
(Supplementary material), 3MB

pnas.1606610113.sm02.mpg
(Supplementary material), 2MB

pnas.1606610113.sm03.mpg
(Supplementary material), 2MB

Citation

Ganguly, A., Manahan, C. C., Top, D., Yee, E. F., Lin, C., Young, M. W., et al. (2016). Changes in active site histidine hydrogen bonding trigger cryptochrome activation. Proceedings of the National Academy of Sciences of the United States of America, 113(36), 10073-10078. doi:10.1073/pnas.1606610113.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002B-9C3C-1
Abstract
Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.