5mC interactions in PDB entry 6MG3 auto-curated with SNAP

Last updated on 2019-09-30 by Xiang-Jun Lu <xiangjun@x3dna.org>. The block schematics were created with DSSR and rendered using PyMOL.

Summary information and primary citation [schematics · contacts · top · homepage · tutorial]

PDB-id

6MG3

Class

transcription-DNA

Method

X-ray (2.05 Å)

Summary

V285a mutant of the c-terminal bzip domain of human c-ebpbeta with 16bp methylated oligonucleotide containing consensus recognition sequence
List of 4 5mC-amino acid contacts:

C.5CM8: stacking-with-A.ARG289 is-WC-paired is-in-duplex [+]:GcG/cGc

C.5CM10: hydrophobic-with-A.ALA285 is-WC-paired is-in-duplex [+]:GcA/TGc

D.5CM108: other-contacts is-WC-paired is-in-duplex [-]:cGc/GcG

D.5CM110: hydrophobic-with-B.ALA285 is-WC-paired is-in-duplex [-]:TGc/GcA

direct SNAP output · DNAproDB 2.0

Reference

Yang, J., Horton, J.R., Wang, D., Ren, R., Li, J., Sun, D., Huang, Y., Zhang, X., Blumenthal, R.M., Cheng, X.: (2019) "Structural basis for effects of CpA modifications on C/EBP beta binding of DNA." Nucleic Acids Res., 47, 1774-1785.

Abstract

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPβ binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPβ DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPβ. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

Base-block schematics in six views [summary · contacts · top · homepage · tutorial]

The 5-methylcytosine group (PDB ligand '5CM') is shown in space-filling model, with the methyl-carbon atom in black.
Watson-Crick base pairs are represented as long rectangular blocks with the minor-groove edge in black. Color code: A-T red, C-G yellow, G-C green, T-A blue.
Protein is shown as cartoon in purple. DNA backbones are shown ribbon, colored code by chain identifier.
The block schematics were created with 3DNA-DSSR, and images were rendered using PyMOL.
Download the PyMOL session file corresponding to the top-left image in the following panel.

List of 4 5mC-amino acid contacts [summary · schematics · top · homepage · tutorial]

The contacts include paired nucleotides (mostly a G in G-C pairing), and amino-acids within a 4.5-A distance cutoff to the base atoms of 5mC.
The structure is oriented in the 'standard' base reference frame of 5mC, allowing for easy comparison and direct superimposition between entries.
The black sphere (•) denotes the 5-methyl carbon atom in 5mC.

	No. 1 C.5CM8: download PDB file for the 5mC entry stacking-with-A.ARG289 is-WC-paired is-in-duplex [+]:GcG/cGc
	No. 2 C.5CM10: download PDB file for the 5mC entry hydrophobic-with-A.ALA285 is-WC-paired is-in-duplex [+]:GcA/TGc
	No. 3 D.5CM108: download PDB file for the 5mC entry other-contacts is-WC-paired is-in-duplex [-]:cGc/GcG
	No. 4 D.5CM110: download PDB file for the 5mC entry hydrophobic-with-B.ALA285 is-WC-paired is-in-duplex [-]:TGc/GcA