Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function Nicole Kuceraa,1, Ira Schmalenb, Sven Henniga,1, Rupert Öllingerc, Holger M. Straussd,2, Astrid Grudzieckic, Caroline Wieczoreka,3, Achim Kramerc, and Eva Wolfb,4,5 aMax Planck Institute of Molecular Physiology, Department of Structural Biology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany; bMax Planck Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany; dNanolytics, Gesellschaft für Kolloidanalytik mbH, Am Mühlenberg 11, 14476 Potsdam, Germany; and cLaboratory of Chronobiology, Charité—Universitätsmedizin Berlin, 10098 Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved January 4, 2012 (received for review August 16, 2011) The three PERIOD homologues mPER1, mPER2, and mPER3 consti- the mBMAL1/mCLOCK transcription factor complex. Addition- tute central components of the mammalian circadian clock. They ally, the PAS domains of NPAS2, mCLOCK, and mPER2 have been contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which reportedtobindheme(13–16).Inthecircadianclock,mPER1and mediate homo- and heterodimeric mPER-mPER interactions as well mPER2 proteins are found in large protein complexes, likely estab- as interactions with transcription factors and kinases. Here we pre- lishing multiple interactions via their PAS domains, the central sent crystal structures of PAS domain fragments of mPER1 and CKIε∕δ binding domain and the C-terminal mCRY binding region mPER3 and compare them with the previously reported mPER2 (17–19). structure. The structures reveal homodimers, which are mediated Studies with mPER knockout mice showed that mPER1 and by interactions of the PAS-B β-sheet surface including a highly con- mPER2 are more essential for circadian rhythmicity than mPER3 served tryptophan (Trp448mPER1, Trp419mPER2, Trp359mPER3). mPER1 (20–22). mPER2 appears to positively regulate the expression of homodimers are additionally stabilized by interactions between clock genes (mper1, mper2, mcry1, mbmal1) in vivo (20, 21), BIOCHEMISTRY the PAS-A domains and mPER3 homodimers by an N-terminal possibly by interacting with REV-ERBα and thereby modulating region including a predicted helix-loop-helix motive. We have ver- its effect on mBMAL1 transcription (23). In contrast, mPER1 ified the existence of these homodimer interfaces in solution and knockout leads to decreased peak amounts of mPER2 and inside cells using analytical gel filtration and luciferase complemen- mCRY proteins in the nucleus, suggesting that mPER1 regulates tation assays and quantified their contributions to homodimer their stability and/or nuclear entry through protein-protein inter- stability by analytical ultracentrifugation. We also show by fluor- actions (20). The subtle effect of mPER3 deficiency on circadian escence recovery after photobleaching analyses that destabiliza- behavior implies that mPER3 is more important for output func- tion of the PAS-B/tryptophan dimer interface leads to a faster tions. Indeed, mPER3 interacts with the nuclear receptor PPAR-γ mobility of mPER2 containing complexes in human U2OS cells. via an N-terminal region including both PAS domains and a pre- Our study reveals structural and quantitative differences between ceding predicted helix-loop-helix motive (24). This interaction the homodimeric interactions of the three mouse PERIOD homolo- represses the PPAR-γ activity and thereby inhibits the adipogen- gues, which are likely to contribute to their distinct clock functions. esis of mesenchymal stem cells. The importance of the per3 gene is also shown by its implication in the delayed sleep phase ∣ ∣ ∣ circadian clock PAS domains PERIOD proteins protein interactions syndrome and the morning or evening sleep timing preferences observed in human populations (25, 26). n mammalians many physiological, behavioral, and biochemical To provide insights into the molecular mechanisms underlying Iprocesses are regulated in a day-time dependent (circadian) the distinct functions and molecular interactions of the three manner. The approximately 24 h period is generated by a circa- mPER homologues, we have solved crystal structures of the PAS dian clock, which is operated by molecular feedback loops. In the domain regions of mPER1 and mPER3 and compared them with main feedback loop, the bHLH (basic-helix-loop-helix)-PAS our mPER2 structure (27). In addition to a conserved PAS-B/ (PER-ARNT-SIM) transcription factors mBMAL1/2, mCLOCK, tryptophan dimer interface, mPER1- and mPER3 homodimers and NPAS2 activate the expression of three PERIOD proteins (mPER1, mPER2, and mPER3) as well as two cryptochromes, mCRY1 and mCRY2. The mPER and mCRY proteins inhibit Author contributions: N.K., I.S., S.H., R.Ö., H.M.S., A.K., and E.W. designed research; N.K., their own transcription, completing the circle of negative feed- I.S., S.H., R.Ö., H.M.S., A.G., and C.W. performed research; A.G. and C.W. contributed new reagents/analytic tools; N.K., I.S., S.H., R.Ö., H.M.S., A.K., and E.W. analyzed data; and N.K. back. The daily regulated expression of mBMAL1 is ensured by and E.W. wrote the paper. a stabilizing feedback loop, in which the heme binding nuclear The authors declare no conflict of interest. receptor REV-ERBα/β represses mBMAL1 expression, whereas This article is a PNAS Direct Submission. ROR-α/β activates it (1, 2). Recently, nontranscriptional circadian Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, oscillations of peroxiredoxin oxidation-reduction, hemoglobin www.pdb.org [PDB ID codes 4DJ2 (mPER1) and 4DJ3 (mPER3)]. dimer-tetramer transitions, and NADH/NADPH oscillations have 1Present address: Western Australian Institute for Medical Research Rear 50 Murray Street been described in human red blood cells, which are interconnected Perth, WA 6000 Australia. with the transcriptional feedback loops in nucleated cells (3, 4). 2Present address: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. The PERIOD proteins and the transcription factors mBMAL1/ 3Present address: Max Planck Institute for Neurological Research, Gleuelerstr. 50, 50931 2, mCLOCK, and NPAS2 contain two tandemly organized PAS do- Cologne, Germany. mains (PAS-A and PAS-B). The PAS domains mediate homo- and 4Present address: Adolf-Butenandt-Institute, Department of Physiological Chemistry, heterodimeric interactions between the mPER homologues (5–8) Butenandtstrasse. 5, 81377 Munich, Germany. as well as interactions of the mPERs with mBMAL1/2, mCLOCK, 5To whom correspondence should be addressed. E-mail: [email protected]. – and NPAS2 (9 12). These interactions regulate the stability and This article contains supporting information online at www.pnas.org/lookup/suppl/ cellular localization of the mPERs and modulate the activity of doi:10.1073/pnas.1113280109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1113280109 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 26, 2021 are stabilized by interfaces located within the PAS-A domain and Ta b l e S 2 ). The mPER homodimers are stabilized by interac- (mPER1) or a predicted helix-loop-helix region N-terminal to tions between the antiparallel PAS-B β-sheet surfaces. Central to 448 the PAS-A domain (mPER3). These additional interfaces lead this interface are the conserved tryptophans Trp mPER1 and 359 419 to increased affinities compared to the mPER2 PAS domain Trp mPER3 [corresponding to Trp mPER2; (27)] as well as 444 ∕ 355 ¼ 415 homodimers. We also provide evidence that the PAS domains of two phenylalanines, Phe mPER1 Phe mPER3 ( Phe mPER2) mPER1 and mPER3 might be able to bind heme. Furthermore, 454 ∕ 365 ð¼ 425 Þ A D and Phe mPER1 Phe mPER3 Phe mPER2 (Fig. 1 and , we show by luciferase complementation assays that the mPER Figs. S2A and S3C). homodimers observed in our crystal structures are also formed in The mPER2 homodimer also involved interactions of the PAS- HEK293 cells. Moreover, our fluorescence recovery after photo- A domain with helix αE and the PAS-B domain of the dimerizing bleaching (FRAP) analyses reveal that destabilization of the PAS- molecule (27) (Fig. S2B). These interactions are not observed B/tryptophan dimer interface generates faster moving mPER2 in the mPER1 and mPER3 structures because the relative orien- containing complexes in human U2OS cells. tation of the monomers is changed in these two homologues A Results (Fig. S3 ; Table S2). Instead, a second homodimer interface is observed in the PAS-A domains of mPER1 and mPER3, which Crystal Structures of Mouse PERIOD1 and Mouse PERIOD3. We have α determined crystal structures of the fragments mPER1[191–502] is mostly mediated by contacts between their antiparallel C B E B and mPER3[108–411], which include the two PAS domains helices (Fig. 1 and , Fig. S2 ). Central to this interface is a 267 179 (PAS-A and PAS-B), the αE helix C-terminal to PAS-B and about tyrosine residue, Tyr mPER1 and Tyr mPER3. In mPER1, the 25 residues N-terminal to PAS-A (Fig. 1, Fig. S1, and Table S1). presence of two glycine residues (Gly264 and Gly268) allows for Both structures revealed noncrystallographic homodimers a close approach of the two αC helices (Fig. 1B). In mPER3, the (Fig. 1 A and D). Each monomer contains two canonical PAS do- αC helices do not approach each other as closely as in mPER1, 264 mains with a five-stranded antiparallel β-sheet (βA-βE) covered on likely due to the exchange of Gly mPER1 to a bulky arginine α α α α α A D 176 E B one face by -helices ( A, A*, B, C) (Fig. 1 and , Fig. S1, (Arg mPER3) (Fig. 1 , Fig. S2 ). A molecule 1 B C PAS-A PAS-B C`* Pro272 Gln237 C` A’ B’ C’ Thr271 E` Thr213 Thr219 Gly268 Tyr267 D’ N Ser221 A B A’* Thr209 A’ E D C C E B D B’ 3.6 Tyr233 E C Leu202 Leu205 Gly264 Ile208 Trp278 N Thr271 Trp448 Tyr200 Ile325 E Pro260 Leu338 N N molecule 2 PAS-B D molecule 1 E F Lys127 Glu252 PAS-A D’ A’ PAS-B E A’ 3.79 D A D C C` Thr131 E` B’ B 3.91 N B C’ 4.55 C His124 Arg227 E 8.44 B’ E Thr118 Tyr179 Leu250 His145 Leu120 Leu248 Arg176 3.73 C Trp190 Thr182 N 2.77 Tyr112 Trp359 N PAS-B molecule 2 Fig.