Struktur-Funktions-Analysen der mCRY-mPER2 und mCRY-mBMAL1 Komplexe

• Antragstellerin: Professorin Dr. Eva Wolf
• Fachliche Zuordnung: Strukturbiologie ; Biochemie
• Förderung: Förderung von 2013 bis 2017
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 246768937

• Erstellungsjahr: 2018

Zusammenfassung der Projektergebnisse

Within this project we successfully determined the crystal structure of the CRY1-PER2 complex and showed that the stability of this complex is regulated by CRY1 disulfide bond formation and Zinc coordination in vitro and in vivo. We also obtained first evidence that CRY1 and the CRY1-PER2 complex bind FAD with a µM affinity and that CRY1-bound FAD is able to undergo redox changes. Our FAD-bound CRY1-PER2 crystal structures reveal partial occupation of the FAD binding pocket reminiscent of binding of the KL001 compound and in support of the multiple conformations of FAD bound to vertebrate CRYs. Our results suggest that cellular redox changes could affect CRY1/PER2 activity in the circadian clock, possibly via disulfide bond formation, Zinc binding or FAD redox changes and potentially also within the multi-subunit complex repressing BMAL1/CLOCK in the late repressive state. In contrast to the high (nM) affinity CRY1-PER2 complex, the CRY1-BMAL1 complex is of much lower (µM) affinity. Within the time frame of this proposal, we purified a CRY1-BMAL1 complex with the acetyl-mimetic K537Q mutation. As this complex did not crystallize, we also purified smaller BMAL1 fragments that still contain K537 or the K537Q mutation for future CRY interaction-, complex formation- and co-crystallization studies. Additionally, we established the purification of mg amounts of full-length CRY2. Furthermore, we performed the cloning and first E.coli expression trials for the K537-acetylated BMAL(490-625). Hence, we have now the tools in hand to expand on the CRY1-BMAL1 and CRY2-BMAL1 cocrystallization as well as SAXS and MS crosslink analyses of CRY1/2-BMAL1 complexes, which we plan to pursue as next steps.

Projektbezogene Publikationen (Auswahl)

• (2014). Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. Cell 157, 1203–1215
  Schmalen I, Reischl S, Wallach T, Klemz R, Grudziecki A, Prabu JR, Benda C, Kramer A, Wolf E
  (Siehe online unter https://doi.org/10.1016/j.cell.2014.03.057)
• „Structural and biochemical examination of the PER1‐PAS domains and the CRY1-PER2 complex as regulatory elements of the mammalian circadian clock“, 2014, LMU Munich
  Ira Schmalen