1. Crystal Structure of Tetrameric Arabidopsis MYC2 Reveals the Mechanism of Enhanced Interaction with DNA 
Teng-fei Lian, Yong-ping Xu, Lan-fen Li, Xiao-Dong Su 
Cell Reports 
Volume 19, Issue 7, Pages (May 2017)
DOI: /j.celrep
Copyright © 2017 The Authors Terms and Conditions

2. Cell Reports 2017 19, 1334-1342DOI: (10.1016/j.celrep.2017.04.057)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1 Structure of the MYC2 bHLH-DNA Complex
(A) Simplified schematic diagram of MYC2. The N-terminal domains were determined by sequence alignment with MYC3.
(B) Multiple sequence alignment of the bHLH domains among TFs, including MYC2-4 in plants and human MYC, MAX, and MAD. Identical residues are labeled with red boxes and residues involved in MYC2 dimer formation are labeled with yellow boxes. Residues that induce specific DNA recognition by MYC2 are labeled with blue triangles, while residues at the MYC2 tetramer interface are labeled with black triangles.
(C) Overview of the MYC2 bHLH-DNA complex crystal structure in two orientations (rotated 90° along the indicated axis). The four monomers in one tetramer are labeled with different colors, and the two α helices in one monomer are named α1 and α2.
See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 2 Interactions for Oligomer Formation
(A) The two parts of residues involved in tetramer formation with key residue numbers labeled.
(B) The two parts of interactions induced dimer formation with key residue numbers labeled. Dotted lines represent for hydrogen bonds or electrostatic interactions in both (A) and (B).
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 3 Different Multimeric States of MYC2 and MYC3
(A) MALS data for the MBP-MYC2 and MBP-MYC3 full length proteins. Calculated MW for MBP-MYC2 is 423 kDa, and MW for MBP-MYC3 is 252 kDa. Superdex 200 column was used. Full lines represent for the UV absorbance at 280 nm, and dotted lines indicate the calculated molecular weight distribution.
(B) MALS data for the MYC2 and MYC3 bHLH domains. Calculated MW of MYC2-bHLH is 39 kDa, and MW of MYC3-bHLH is 22 kDa. Superdex 75 column was used. Full lines represent for the UV absorbance at 280 nm, and dotted lines indicate the calculated molecular weight distribution.
(C) Size exclusion chromatography (Superdex75) for the MYC2 and MYC3 bHLH domains.
(D) Size exclusion chromatography (Superdex75) for the MYC2 and MYC3 bHLH-DNA complexes. Absorbance at 280 nm are shown as full lines, while absorbance at 260 nm are shown as dotted lines.
(E) ITC result for MYC2 bHLH domain titration with G-box DNA. The error bars represent the SD from three individual experiments.
(F) ITC result for MYC3 bHLH domain titration with G-box DNA.
Cyan lines represent the results for the MYC2 proteins, whereas blue lines represent the results for MYC3 proteins.
See also Figures S2 and S3.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 4 MYC2 Tetramers Enhance DNA Binding and Gene Regulation
(A) Structural analysis between the MYC2 dimer and DNA recognized by another dimer in the same tetramer. Electrostatic interactions are shown as dotted lines.
(B) ITC result for the MYC2-dimer mutant with the G-box DNA.
(C) ITC result for MYC2-mutweak with the G-box DNA.
(D) Dual luciferase assays for MYC2 overexpression, MYC2-dimer mutant overexpression, MYC3 overexpression protoplasts and wild-type (WT) protoplasts with no protein overexpression. The results from the assays using the jaz2 and ebf1 gene promoters before luciferase are shown as gray and black columns, respectively. The relative luciferase activity of each system was presented as a percentage of Renilla luciferase intensity. Error bars denote the SD from three individual experiments. The p value was calculated with the Student’s t test. ∗∗p < 0.01 and ∗∗∗p <
(E) Western blot assay shows that the protein expression levels of MYC2, MYC2-dimer mutant, and MYC3 were similar in protoplasts. All proteins were expressed with 3x FLAG tag in C-terminal.
(F) FRET assay suggests that MYC2 induce DNA looping. Spacer DNA is defined as the DNA fragment between two G-box sites (Figure S4B). Blue columns represent for the fluorescence intensity of DNA, orange columns represent for the fluorescence intensity of DNA-MYC3 complex, and the fluorescence intensity of DNA-MYC2 complex are shown as purple columns. Error bars denote the SD from three individual experiments.
See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions