Figure 4. The secondary pocket is involved in CRY-CLOCK-BMAL1 complex assembly and repression.
(A) Relative positions of the two large pockets on CRY2. (B) Surface representation of CRY2 with side chains of the serine loop shown in sticks. PER2 α1 helix inserts into a hydrophobic cleft. Compared to other CRY2 complexed forms, the serine loop flips up and engages PER2. (C) The serine loop lies opposite to the CRY α4 helix, which together frame the secondary pocket, the α4 helix contains three residues (CRY1 G106R and R109Q, CRY2 E121K), whose mutations result in a weak repression phenotype. (D–F) Co-immunoprecipitation assays show that the CRY1 R109Q mutant is unable to bind CLOCK-BMAL1, but retains PER1 and PER2 binding.
Figure 4—figure supplement 1. Locations of structurally plastic loops on CRY2.
(A) The phosphate-binding loop and interface loop frame opposite sides of the FAD-binding pocket. Superimposition analysis shows the FAD-binding pocket as a regulatory hotspot, which can bind metabolic cofactor, FAD (yellow), clock-modulating small molecule, KL001 (cyan), and FBXL3 C-terminal tail (green). (B) Locations of CRY2 loops; interface loop (yellow), phosphate-binding loop (red), serine loop (purple), and protrusion loop (light blue), which in light-sensitive CRYs occludes part of the FAD-binding pocket but is pushed outward and maintains an open FAD-binding pocket in vertebrate CRYs.
Figure 4—figure supplement 2. CRY-PHR superposition: including CRY1 apo (red), CRY2 apo (light blue), KL001-bound (green), FAD-bound (orange), FBXL3-bound (cyan), and PER2-CBD-bound (gray) CRY.
(A) Serine loop undergoes a large conformational change after PER2-CBD binding. (B and C) The interface loop and phosphate-binding loop are also sites of high structural plasticity. (D) Overall CRY-PHR showing the global structure adopts a common fold.
Figure 4—figure supplement 3. Major differences between CRY1-PER2-CBD and CRY2-PER2-CBD complex structures.
Superposition of the two structures reveals major structural dissimilarities between the two paralogs at the CRY secondary pocket and a residual fusion-protein sequence (yellow) in CRY1-bound PER2-CBD. The PER2-CBD (dark blue) N-terminus together with the artifactual sequence (AGLEVLFQGPDSM) forms a β-hairpin and induces an inward conformation of the CRY1 (light green) serine loop.