title: Changes in active site histidine hydrogen bonding trigger cryptochrome activation.
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.
authors.affiliation: Max-Planck-Institut für Kohlenforschung, 45470 Mülheim an der Ruhr, Germany;
authors.forename: Abir
authors.initials: A
authors.lastname: Ganguly
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
authors.forename: Craig C
authors.initials: CC
authors.lastname: Manahan
authors.affiliation: Laboratory of Genetics, The Rockefeller University, New York, NY 10065.
authors.forename: Deniz
authors.initials: D
authors.lastname: Top
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
authors.forename: Estella F
authors.initials: EF
authors.lastname: Yee
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
authors.forename: Changfan
authors.initials: C
authors.lastname: Lin
authors.affiliation: Laboratory of Genetics, The Rockefeller University, New York, NY 10065.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Young
authors.affiliation: Max-Planck-Institut für Kohlenforschung, 45470 Mülheim an der Ruhr, Germany;
authors.forename: Walter
authors.initials: W
authors.lastname: Thiel
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; bc69@cornell.edu.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Crane
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D057906:Circadian Clocks / Q000235:genetics*; D056931:Cryptochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008027:Light; D056004:Molecular Dynamics Simulation; D010084:Oxidation-Reduction; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005415:Flavins; D011522:Protons; D011994:Recombinant Proteins; C119828:cry protein, Drosophila; C096445:tim protein, Drosophila; C025232:semiquinone radicals; D006639:Histidine
keywords: flavoprotein; light sensing; molecular dynamics; photochemistry; redox