The secondary pocket of cryptochrome 2 is important for the regulation of its stability and localization

Parlak G. C., Camur B. B., Gul Ş., Ozcan O., Baris I., Kavakli I. H.

Journal of Biological Chemistry, vol.298, no.9, 2022 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 298 Issue: 9
  • Publication Date: 2022
  • Doi Number: 10.1016/j.jbc.2022.102334
  • Journal Name: Journal of Biological Chemistry
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Applied Science & Technology Source, Aquatic Science & Fisheries Abstracts (ASFA), Artic & Antarctic Regions, BIOSIS, CAB Abstracts, Chemical Abstracts Core, Compendex, EMBASE, MEDLINE, Veterinary Science Database, Directory of Open Access Journals
  • Keywords: circadian clock, cryptochrome, nuclear transport, protein stability, SNP
  • Bezmialem Vakıf University Affiliated: No


Human clock-gene variations contribute to the phenotypic differences observed in various behavioral and physiological processes, such as diurnal preference, sleep, metabolism, mood regulation, addiction, and fertility. However, little is known about the possible effects of identified variations at the molecular level. In this study, we performed a functional characterization at the cellular level of rare cryptochrome 2 (CRY2) missense variations that were identified from the Ensembl database. Our structural studies revealed that three variations (p.Pro123Leu, p.Asp406His, and p.Ser410Ile) are located at the rim of the secondary pocket of CRY2. We show that these variants were unable to repress CLOCK (circadian locomotor output cycles kaput)/BMAL1 (brain and muscle ARNT-like-1)–driven transcription in a cell-based reporter assay and had reduced affinity to CLOCK–BMAL1. Furthermore, our biochemical studies indicated that the variants were less stable than the WT CRY2, which could be rescued in the presence of period 2 (PER2), another core clock protein. Finally, we found that these variants were unable to properly localize to the nucleus and thereby were unable to rescue the circadian rhythm in a Cry1−/−Cry2−/− double KO mouse embryonic fibroblast cell line. Collectively, our data suggest that the rim of the secondary pocket of CRY2 plays a significant role in its nuclear localization independently of PER2 and in the intact circadian rhythm at the cellular level.