Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4

Abstract

PC4, a well-known general transcription cofactor, has multiple functions in transcription and DNA repair. Residue W89, is engaged in stacking interactions with DNA in PC4, but substituted by tyrosine in some PC4 orthologous proteins. In order to understand the consequences and reveal the molecular details of this substitution we have determined the crystal structures of the PC4 orthologue MoSub1 and a PC4 W89Y mutant in complex with DNA. In the structure of MoSub1-DNA complex, Y74 interacts directly with a single nucleotide of oligo DNA. By comparison, the equivalent residue, W89 in wild type PC4 interacts with two nucleotides and the base of the second nucleotide has distinct orientation relative to that of the first one. A hydrophobic patch around W89 that favours interaction with two nucleotides is not formed in the PC4 W89Y mutant. Therefore, the change of the surface hydrophobicity around residue 89 results in a difference between the modes of DNA interaction. These results indicate that the conserved Y74 in MoSub1 or W89 in PC4, are not only key residues in making specific interactions with DNA but also required to determine the DNA binding mode of PC4 proteins.

The effect of phosphate ion on the ssDNA binding mode of MoSub1, a Sub1/PC4 homolog from rice blast fungus

“…This DNA conformation differed from that of the PC4–ssDNA complex, in which the ssDNA bent back to interact with the other monomer of the same dimer and formed a hairpin‐like structure (Figure D). This structure was also different from that of the previously solved MoSub1–ssDNA structure, in which the ssDNA chain extended straight to interact with another monomer from a dimer in an ASU (Figure C).…”

“…The crystal structure of MoSub1–ssDNA in phosphate buffer was solved by molecular replacement at a resolution of 2.04 Å. The new crystal belongs to I121 ( a = 42.01 Å, b = 80.19 Å, c = 76.91 Å; α = γ = 90.0°, β = 91.88°), and two MoSub1 molecules and one ssDNA chain were contained in an asymmetric unit (ASU), which is different from the previous crystal belonging to P12 1 1 ( a = 84.2 Å, b = 58.0 Å, c = 83.8 Å; α = 90.0°, β = 107.0°, γ = 90.0°) with six protein monomers and three ssDNA molecules in an ASU . Residues 34‐116 of each monomer and 12 nucleotides of DNA can be seen clearly in the electron density map and built in the model.…”

“…This DNA binding mode can be recognized as the U mode. More recently, the crystal structure of the MoSub1–ssDNA complex was determined, in which ssDNA extended straight and linked two neighboring MoSub1 dimer molecules (Figure C) . This ssDNA binding status can be termed the straight mode.…”

“…1,8 MoSub1, a Sub1 homolog from rice blast fungus (Magnaporthe oryzae), also binds ssDNA tightly, and the crystal structures of MoSub1 and its complex with ssDNA have also been determined. 15,16 These structures show high similarity to the PC4 structure and they have a similar dimer interface and DNA-binding region, but with some differences in ssDNA conformation.…”

“…Sub1, which differs from PC4 in domain structure, particularly the presence of a C‐terminal extension, and its interaction with TFIIB but not TFIIA, shares very high homology in the DNA binding region . MoSub1, a Sub1 homolog from rice blast fungus ( Magnaporthe oryzae ), also binds ssDNA tightly, and the crystal structures of MoSub1 and its complex with ssDNA have also been determined . These structures show high similarity to the PC4 structure and they have a similar dimer interface and DNA‐binding region, but with some differences in ssDNA conformation.…”