Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains

Abstract

The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB–DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2–DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2–DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB–DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi

“…The first solved SBF/MBF crystal structure, Mbp1 from S. cerevisiae in the absence of DNA, suggested Mbp1 recognizes its MCB (Mlu I cell cycle box, ACGCGT) binding site via a recognition helix (Taylor et al, 1997; Xu et al, 1997). However, a recent crystal structure of PCG2, an SBF/MBF homolog in the rice blast fungus Magnaporthe oryzae , in complex with its MCB binding site does not support this proposed mode of DNA binding (Liu et al, 2015). In striking contrast to many wHTH structures, in which the recognition helix is the mediator of DNA binding specificity, the wing of PCG2 binds to the minor groove to recognize the MCB binding site.…”

The Inner Nuclear Membrane Protein Bqt4 in Fission Yeast Contains a DNA-Binding Domain Essential for Telomere Association with the Nuclear Envelope

“…Comparing with Mbp1-DNA interaction, Bqt4 has different DNA-binding characteristics, which could be attributed to the structural difference between Bqt4 and Mbp1. The K d of Bqt4 2-180 for dsDNA (20 bp) was $10 mM, which is $10 times weaker than the MoMbp1-dsDNA interaction (K d = 0.81 mM) (Liu et al, 2015). This relatively low DNA-binding affinity can be explained by the sequence variation in the recognition helix.…”

“…The optimal DNA-binding activity requires an extra C-terminal loop of Bqt4 NTD . This is not surprising, since the dynamically disordered C terminus of the DNA-binding domain of Mbp1 has also been reported to be crucial for the DNA-protein interaction (Liu et al, 2015;Taylor et al, 2000). Comparing with Mbp1-DNA interaction, Bqt4 has different DNA-binding characteristics, which could be attributed to the structural difference between Bqt4 and Mbp1.…”

“…The sequence and structural disparities are evident in the connecting loop regions. The most divergent loop is the wing loop of the wHTH motif located between a3 and b5, which is involved in specific DNA recognition (Figures 2A and S2A) (Liu et al, 2015). It indicates that Bqt4 may have different DNA-binding features.…”

“…Bqt4 NTD Is an Atypical APSES-like Domain An unbiased search for structurally homologous proteins using the Dali server (Holm and Rosenstrom, 2010) revealed that the structure of Bqt4 NTD is highly similar to the fungal APSES domains of a group of transcription factors, including Saccharomyces cerevisiae Mbp1 (Xu et al, 1997), Magnaporthe oryzae Mbp1 (Liu et al, 2015), and S. cerevisiae Swi6 (Taylor et al, 2010) (Figure 2A). The common core region of Bqt4 NTD (33-140) can be superimposed to ScMbp1, MoMbp1, and ScSwi6 with root-mean-square deviation values of 1.9, 2.1, and 3.0 Å for 94, 96, 87 equivalent C a atoms, respectively ( Figure 2A).…”