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. 2009 Apr 28;7(4):e94.
doi: 10.1371/journal.pbio.1000094.

Structural and Functional Analyses of PAS Domain Interactions of the Clock Proteins Drosophila PERIOD and Mouse PERIOD2

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Free PMC article

Structural and Functional Analyses of PAS Domain Interactions of the Clock Proteins Drosophila PERIOD and Mouse PERIOD2

Sven Hennig et al. PLoS Biol. .
Free PMC article

Abstract

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Domain Architecture and Sequence Alignment of dPER and Mouse PERIOD Proteins
(A) Domain architecture of dPER and mouse PERIOD2 (mPER2). The two PAS domains (PAS-A and PAS-B); the cytoplasmic localization domain (CLD, orange bar); nuclear localization signals (NLS, red bars); NES (green bars); the conserved C-domain; the threonine-glycine (TG) repeat region; and the dCLK:CYC inhibition domain (CCID) of dPER and/or mPER2 are shown schematically. CKIɛ, mCRY1/2, dTIM, and DBT are depicted at their binding sites. The dPER and mPER2 fragments used for crystallization and biochemical studies are represented as black bars. (B) Sequence alignment of the PAS-A and PAS-B domains as well as helices αE and αF of dPER (Swissprot accession number P07663) with mPER 1, 2, and 3 (Swissprot accession number [1] O35973, [2] O54943, and [3] O70361). Secondary structure elements were assigned from the dPER[232–599] structure ([31], PAS-A, βA → βE; PAS-B, βA′→ βE′). Partially disordered insert regions are depicted as yellow waves. The sequence alignment was generated in ClustalW [78]. dPER residues Trp482, Met560, Glu474, His492, Arg494 mutated herein as well as the sites of the per L long period mutation V243D and the per S/T short period mutations S589D and G593D are highlighted by cyan arrows, mutated mPER2 residues Trp419, Ile427, and Phe415 by dark blue arrows. dPER residues in the Trp482 binding pocket of PAS-A are marked with cyan stars (*), dPER residues interacting with the αF helix with cyan open circles (°). Residues in the dimer interfaces of mPER2 are marked with dark blue stars (*, PAS-B β-sheet interface) or hashes (#, PAS-A dimer interactions). Residues involved in intramolecular PAS domain interactions are marked with cyan (dPER) and dark blue (mPER2) plus signs (+). S/M, short-mutable region.
Figure 2
Figure 2. Crystal Structures of Drosophila PERIOD
(A) Ribbon presentation of the dPER[232–599] dimer with molecule 1 shown in cyan, molecule 2 in grey. The inset shows a close-up view highlighting residues Trp482 and Met560, which have been mutated in this study. The per L mutation site (Val243) is also highlighted. (B) Stereo view of the dPERΔαF[232–538] monomer structure. (C) Superposition of the dPERΔαF[232–538] monomer structure (green) on molecule 1 (cyan) and 2 (grey) of the dPER[232–599] dimer. The two orientations are related by 180 ° rotations.
Figure 3
Figure 3. Analytical Gel Filtration and Ultracentrifugation of dPER
(A) Analytical gel filtration of dPER[232–599] wild-type and mutants dPER(M560D), dPER(W482E), and dPER(M560D/W482E). Same amounts of E. coli expressed and purified proteins were loaded on a HiLoad Superdex 200 16/60 gel filtration column. The elution positions of the monomeric dPER L[232–599]V243D mutant and dPERΔαF[232–538] [31] are indicated. (B) A typical sedimentation equilibrium experiment of dPER[232–599] wild-type at a single concentration (out of four). Data of other fragments and mutants were of comparable quality. (C) Summary table of analytical ultracentrifugation. K D = 1/K A. dof, degrees of freedom; fix, molar mass fixed to the expected value. 95% confidence intervals are given in brackets. * A statistically equivalent description of the data is obtained for a single-ideal species model (rmsd of 1.12 × 10−2) but with a slightly lower molar mass (81.41 kg/mole, (81.22–82.13) kg/mol) than expected for the dimer (83.64 kg/mol).
Figure 4
Figure 4. Structural Analysis of PAS Domain Interactions in mPER2[170–473]
(A) Ribbon presentation of the mPER2[170–473] homodimer with molecule 1 shown in dark blue, molecule 2 in grey. The conserved Trp419 residues are shown as atomic stick figure. (B) Close-up view of the dimer interface formed by antiparallel packing of the PAS-B β-sheet surfaces. Interacting residues are highlighted as atomic stick figure. Residues Trp419, Ile427, and Phe415 have been mutated within this study. Top, molecule 1 in dark blue; bottom, molecule 2 in grey. (C) Close-up view of the dimer interface formed by antiparallel packing of the PAS-B β-sheet surfaces. Residues and water molecules mediating dimer interactions are highlighted as atomic stick figures and red spheres. Pro390, Phe425, and Glu342 have been omitted for clarity. Ser414 and Ile416 establish main chain interactions to water molecules in the interface. The 1 sigma 2fo-fc composite omit map is shown in blue. Similar view as in Figure 4B, but with molecules 1 and 2 switched/rotated around the noncrystallographic symmetry (NCS) axis. Top, molecule 2 in dark blue; bottom, molecule 1 in grey. (D) Close-up view showing dimer interactions of the PAS-A domain with the PAS-B domain and helix αE. Interacting residues are highlighted as atomic stick figure. Pairs of interacting residues (hydrogen bond or van der Waals interactions) are connected by dashed lines.
Figure 5
Figure 5. Structural Comparison of mPER2 and dPER Reveals a Putative NES in dPER
(A) Left, superposition of molecule 1 of mPER2[170–473] (dark blue) and molecule 1 of dPER[232–599] (cyan, αF helix omitted). Largest changes are seen in the αE helix and the preceding linker, the αC′ helix as well as the hydrophobic pocket of PAS-A. Part of the dimerizing dPER[232–599] molecule 2 is shown in cyan. Right, superposition of the PAS-A–Trp482 dimer interface of dPER (cyan) with the PAS-A domain of mPER2 (dark blue), close-up view. His238 of mPER2 and Ala287 of dPER are shown as atomic stick figure. Trp482 of the dimerizing dPER molecule 2 is shown in cyan. Structural changes in strands βA and βB as well as the βA-βB loop are indicated by black arrows. (B) Close-up view of αE of mPER2 including an NES sequence. Residues Leu460, Ile464, Leu467, and Met469 of the NES sequence as well as Val472 (corresponding to Val538 of dPER) are shown as atomic stick figure. The dimerizing mPER2 molecule is shown in grey. (C) Superposition of the αE helix of mPER2 (dark blue) and dPER (cyan). The residues of the putative NES sequence in dPER (Ile526, Ile530, Leu534, Val538) are shown in green as atomic stick figure. Their location is compared to the NES residues Leu460, Ile464, Leu467, and Met469 as well as Val472 of mPER2 (magenta).
Figure 6
Figure 6. Analytical Gel Filtration and Ultracentrifugation of mPER2
(A) Analytical gel filtration of mPER2 interface mutants. Wild-type, W419E, I427E, and F415E mutant versions of mPER2[128–473] were analysed on a HiLoad Superdex 200 10/30 gel filtration column. The elution positions of the crystallized mPER2[170–473] fragment as well as the marker proteins Ovalbumin and Apotransferrin are indicated. (B) A typical sedimentation equilibrium experiment of dPER[128–473] wild-type at a single concentration (out of four). Data of other fragments and mutants were of comparable quality. (C) Summary table of analytical ultracentrifugation of mPER2 variants. K D = 1/K A. dof, degrees of freedom; fix, molar mass fixed to the expected value. 95% confidence intervals are given in brackets.
Figure 7
Figure 7. Trp419Glu Mutation Interferes with mPER2 Homodimerization in HEK293 Cells
HEK293 cells were transfected with pairs of C-terminally V5- and N-terminally HA-tagged mPER2 full-length proteins or mPER2[128–473] fragments either as wild-type or as W419E variants (lane 1, mPER2 wt-V5/HA-mPER2 wt; lane 2, mPER2 W419E-V5/HA-mPER2 W419E; lane 3, mPER2[128–473]wt-V5/HA-mPER2[128–473]wt; lane 4, mPER2[128–473] W419E-V5/HA-mPER2[128–473]W419E). 48 h after transfection cells were lysed in Co-IP buffer. In the upper two panels of the figure the expression of the corresponding pairs of V5- and HA-tagged mPER2-variants was confirmed by SDS-PAGE/Western blotting using anti-V5- and anti-HA-antibodies, respectively. For immunoprecipitation each cell extract was incubated with anti-V5–antibody and G protein-coupled agarose beads. Co-IP of HA-tagged proteins was analyzed by Western blotting using an anti-HA–antibody (third panel, IP:α-V5, WB:α-HA). In the bottom panels the minus anti-V5-antibody control samples are shown. Blot regions at the migration distance of either full-length mPER2 (lanes 1, 2) or the mPER2[128–473] fragment (lanes 3, 4) are depicted. No unspecific binding of PER2 full-length and PER2 fragment proteins to the beads was detected. The weak band observed in the minus anti-V5–antibody control sample of the mPER2 fragment (lanes 3, 4) is not mPER2, as it is also observed in untransfected HEK293-lysates (not shown).
Figure 8
Figure 8. Yeast-Two-Hybrid Analysis of dPER-dTIM Interaction
(A) dPER[232–599] structure with residues mutated within the yeast-two-hybrid study highlighted. (B) Yeast-two-hybrid analysis of the interaction between wild-type or mutant versions of dPERΔαF[232–538] and full-length dTIM. Wild-type dPER[232–538] and wild-type dTIM show a strong interaction (see column 4) that is abolished by the introduction of the triple mutation E474R/H492S/R494D (B3) in the PAS-B domain (see column 5 and arrow).

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