Article Figures & Data
Figures
- Figure 1.
CCT Domain-Containing Proteins from All Three Families Can Be Physically Associated with the GHD8/OsNF-YC2 Dimer.
(A) LCI assay shows the interactions among the full-length CCT domain-containing proteins, GHD8, and OsNF-YC2. For each representation, the constructs used for each infiltration were listed at the left corresponding section. Fluorescence intensities represented their interaction activities. CCT domain-containing proteins (HD1, GHD7, and GHD7.1) and GHD8 were fused to the N- and C-terminal domains of LUCIFERASE (NLUC and CLUC), respectively. OsNF-YC2 was cloned into the third vector pCAMBIA1301s. Co-transfections of CCT-NLUC (HD1, GHD7, or GHD7.1), GHD8-CLUC, and OsNF-YC2 could produce robust luciferase activities, while other infiltrations lacking one or two proteins brought about no or slight signals.
(B) Pull-down assay showed the interactions among the full-length CCT domain-containing proteins, GHD8, and OsNF-YC2. His-tagged CCT protein (HD1, GHD7, or GHD7.1), Myc-tagged GHD8, and StrepII-tagged OsNF-YC2 were co-transfected into Expi293F cells. The supernatant from lysed cells was loaded onto StrepII beads. Both GHD8 and CCT proteins were co-eluted with OsNF-YC2. The proteins were detected by immunoblot with antibodies against StrepII, His, and Myc. ΔCCT indicated deletion of CCT domain.
- Figure 2.
CCT/GHD8/OsNF-YC2 Complex Containing HD1CCT, GHD7CCT, or GHD7.1CCT Can Bind DNA Probe Containing ‘CCACA’ Motif.
(A) SEC analysis shows the interaction between the GHD8/OsNF-YC2 dimer and HD1CCT, GHD7CCT, or GHD7.1CCT. The same eluted fractions of each SEC injection were subjected to SDS-PAGE followed by Coomassie blue staining. CCT domains were fused with an MBP tag at the N terminus. The x axis indicates the elution volume, and the y axis indicates the protein’s absorption at 280 nm. The peaks of GHD8/OsNF-YC2, MBP-tagged CCT domain, and CCT/GHD8/OsNF-YC2 complex are colored red, orange, and green, respectively.
(B) EMSA analysis show that the CCT/GHD8/OsNF-YC2 complex containing HD1CCT, GHD7CCT, and GHD7.1CCT can bind to DNA containing the ‘CCACA’ motif. The DNA comes from the Hd3a promoter (named OsCORE2) and was labeled by FAM at the 5′ end. The final protein concentrations in each set of four lanes (lanes 2 to 5, 6 to 9, 10 to 13, 14 to 17, 18 to 21, 22 to 25, and 26 to 29) were 0.25, 0.50, 1.00, and 2.00 μM, respectively.
(C) ITC analysis of the interaction between DNA and the CCT/GHD8/OsNF-YC2 complex containing HD1CCT, GHD7CCT, or GHD7.1CCT.
- Figure 3.
Overall Structure of the DNA-Bound HD1CCT/GHD8/OsNF-YC2 Complex.
(A) Domain architectures of HD1, GHD8, and OsNF-YC2. The dotted boxes indicate the protein boundaries used for structural determination.
(B) Crystal structure of the DNA-bound HD1CCT/GHD8/OsNF-YC2 heterotrimer. The key secondary structure elements of the HD1CCT are labeled. HD1CCT, GHD8, and OsNF-YC2 are colored green, slate, and salmon, respectively. The DNA OsCORE2 is colored gray with ‘CCACA’ nucleotides highlighted in yellow. Two perpendicular views are presented. All structure figures were prepared with the tool PyMOL.
(C) Electrostatic surface of the GHD8/OsNF-YC2 dimer. Blue, white, and red colors represent positive, neutral, and negative surfaces, respectively. The negative interface of the dimeric GHD8/OsNF-YC2 matches the polar residues in helix α1 of HD1CCT, whereas the positive interface perfectly matches the DNA. The relevant CCT region is shown as cartoon (main chain) and stick (side chains) representations. DNA is shown as gray sticks.
- Figure 4.
Detailed Interactions between the Protein Interaction Element of HD1CCT and GHD8/OsNF-YC2 Dimer.
(A) HD1CCT sequence and the key residues interacting with the GHD8/OsNF-YC2 dimer and DNA. HD1CCT consists of two elements: the PIE and the DRE. The magenta dots indicate the residues involved in protein interactions, and the blue dots indicate the residues interacting with DNA.
(B) Zoom-in of HD1CCT residues interacting with GHD8/OsNF-YC2 dimer. HD1CCT, GHD8, and OsNF-YC2 are colored green, slate, and salmon, respectively.
(C) SEC analysis of the interaction between the mutated HD1CCT (R338A or Y345A) and GHD8/OsNF-YC2 dimer. The same eluted fractions of each SEC injection are subjected to SDS-PAGE followed by Coomassie blue staining. CCT domains are fused with an MBP tag at the N terminus. The x axis indicates the elution volume, and the y axis indicates the protein’s absorption at 280 nm. The peak of the MBP-tagged HD1CCT (R338A or Y345A) is colored orange, and the peak of incubation product of MBP-tagged HD1CCT (R338A or Y345A) and GHD8/OsNF-YC2 is colored green. Mutated HD1CCT failed to comigrate forward with GHD8/OsNF-YC2, indicating the abrogation of HD1CCT/GHD8/OsNF-YC2 trimer formation.
- Figure 5.
Structural Basis of Specific DNA Recognition by the HD1CCT/GHD8/OsNF-YC2 Complex.
(A) Schematic illustration of protein–DNA interactions. Amino acids of HD1CCT, GHD8, and OsNF-YC2 are colored green, slate, and salmon, respectively. Dashed lines indicate H-bonds and salt bridges. The ‘CCACA’ motif is shaded in yellow.
(B) Specific recognition of the ‘CCACA’ motif by HD1CCT residues. The main chain of amino acids is shown as cartoons and the side chains are shown as sticks and colored green. Bases are shown as sticks and colored according to atom type (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). Amino acids and nucleotides are labeled in black and red letters, respectively. All structure figures were prepared with the tool PyMOL.
- Figure 6.
The DNA Binding Activity of the Mutated HD1CCT/GHD8/OsNF-YC2 Complexes Were Measured by EMSA and ITC Assay.
(A) EMSA analysis of the interaction between DNA and HD1CCT/GHD8/OsNF-YC2 complex containing mutated HD1CCT (R363A, R370A, R372A, K374A, R376A, or F377A). All the mutated residues are located in the DRE region of HD1CCT. Protein was added with increasing final concentrations of 0.25, 0.50, 1.00, and 2.00 μM in each set of four lanes (lanes 2 to 5, 6 to 9, 10 to 13, 14 to 17, 18 to 21, 22 to 25, and 26 to 29).
(B) ITC analysis of the interaction between DNA and the mutated HD1CCT/GHD8/OsNF-YC2. The DNA sequence used for EMSA and ITC is 5′-CTCACTCTCAACCACAGCTCGAT-3, similar to that in Figure 2B.
- Figure 7.
Structural Comparison of DNA-Bound HD1CCT/GHD8/OsNF-YC2 and Human NF-Y Complex.
(A) Sequence alignment of CCT domain and DNA binding domain of NF-YA. CCT domains come from HD1 (O. sativa, LOC_Os06g16370), CO (Arabidopsis, At5g15840), EgCOL2 (Eucalyptus grandis, XP_010029446.1), CrCO (Chlamydomonas reinhardtii, Cre06.g278159), and PpCOL1 (Physcomitrella patens, XP_001783619.1). NF-YA proteins are from OsNF-YA7 (O. sativa, XP_015631822.1), HsNF-YA (Homo sapiens, NP_002496.1), CeNF-YA (Caenorhabditis elegans, NP_509999.1), HAP2 (Saccharomyces cerevisiae, NP_011277.1), and DmNF-YA (Drosophila melanogaster, NP_648313.1). The alignment was performed by the Clustal Omega website service (https://www.ebi.ac.uk/Tools/msa/clustalo/). Secondary structural elements are shown above. Sequence identity is indicated in white letters against a red background, and sequence similarity is shown in red letters. Loop 1 was highlighted by gray background.
(B) Structural comparison of DNA-bound HD1CCT/GHD8/OsNF-YC2 and human NF-Y complex (PDB:4AWL). Middle representation: overall superposition of DNA-bound HD1CCT/GHD8/OsNF-YC2 and human NF-Y complex (PDB:4AWL). HD1CCT and HsNF-YA are shown as cartoons and colored green and magenta, respectively. NF-YB/YC are shown as surface representation. GHD8 and HsNF-YB are colored in slate. OsNF-YC2 and HsNF-YC are colored in salmon. Both DNAs are shown as cartoon representation and are respectively colored wheat and gray. Left inset exhibits the enlarged image showing the detailed interactions between HD1CCT loop 1 and GHD8/OsNF-YC2. Right inset displays the enlarged image showing the detailed interactions between of HsNF-YA loop1 and HsNF-YB/YC.
(C) Superposition of the DREs of HD1CCT and HsNF-YA. HD1CCT and HsNF-YA are colored in green and magenta, respectively.
(D) Comparison of the side chains of residues interacting with DNA in HD1CCT and HsNF-YA. Partial DNA sequences recognized by HD1CCT and HsNF-YA are listed at left. The DNA bases are consecutively numbered from 1 at the 5′ end, and DNA bases on the complementary chain are numbered according to its partner starting with 1′. The core motifs ‘CCACA’ and ‘CCAAT’ are colored red and the flanking bases are colored black. Top representation shows zoom-in of residues of HD1CCT, and bottom representation shows zoom-in of residues of HsNF-YA. In the zoom-in images, amino acids are indicated as black regular letters followed by the numbers indicating their positions. The DNA bases are indicated in gray italic letters followed by the numbers indicating their positions.
Tables
Parameter DNA-bound HD1CCT/GHD8/OsNF-YC2 GHD8/OsNF-YC2 Space group P 31 2 1 P 61 Cell dimensions a, b, c (Å) 136.08, 136.08, 62.96 89.27, 89.27, 81.34 α, β, γ (°) 90, 90, 120 90, 90, 120 Wavelength (Å) 0.97914 0.97946 Resolution (Å) 45 ∼2.55 (2.66∼2.55) 45∼2.00 (2.05∼2.00) Rmerge (%) 9.0 (80.0) 8.2 (58.0) Rpim (%) 2.9 (26.7) 2.6 (18.1) I/σ 14.8 (2.4) 21.2(5.3) Completeness (%) 99.5 (99.2) 99.7 (98.0) Number of measured reflections 217, 423 (24,970) 270, 613 (19,817) Number of unique reflections 22, 041 (2,658) 25, 020 (1,825) Redundancy 9.9 (9.4) 10.8 (10.9) Wilson B factor (Å2) 45.5 22.3 Rwork/Rfree (%) 19.95/22.58 17.06/21.46 Number of atoms Protein 1,708 2,737 Main chain 840 1,356 Side chain 868 1,381 Water molecules 16 168 Other entities (DNA) 1,025 0 All atoms 2,749 2,905 Average B value (Å2) Protein 67.9 36.7 Main chain 64.6 31.9 Side chain 71.1 41.5 Water molecules 63.0 46.3 Other entities (DNA) 77.7 0 All atoms 71.5 37.3 Root mean square deviations Bonds (Å) 0.010 0.007 Angle (°) 1.094 0.853 Ramachandran plot statistics (%) Most favorable 96.1 98.5 Additionally allowed 3.9 1.5 Generously allowed 0 0 Values in parentheses are for the highest resolution shell. Rmerge = ΣhΣi|Ih,i-Ih|/ΣhΣiIh,i, where Ih is the mean intensity of the i observations of symmetry-related reflections of h. R = Σ|Fobs − Fcalc|/ΣFobs, where Fcalc is the calculated protein structure factor from the atomic model (Rfree was calculated with 5% of the reflections selected).
Supplemental Data
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