Identification of the single-stranded DNA binding surface of the transcriptional coactivator PC4 by NMR

The C-terminal domain of the eukaryotic transcriptional cofactor PC4 (PC4(C TD)) is known to bind with nanomolar affinity to single-stranded (ss)DNA. H ere, NMR is used to study DNA binding by this domain in more detail. Amide resonance shifts that were observed in a (HN)-H-1-N-15-HSQC-monitored titra tion of N-15-labeled protein with the oligonucleotide dT(18) indicate that binding of the nucleic acid occurs by means of two anti-parallel channels t hat were previously identified in the PC4(CTD) crystal structure. The beta- sheets and loops that make up these channels exhibit above average flexibil ity in the absence of ssDNA, which is reflected in higher values of T-1 rho , reduced heteronuclear nuclear Overhauser effects and faster deuterium exc hange rates for the amides in this region. Upon ssDNA binding, this excess flexibility is significantly reduced. The binding of ssDNA by symmetry-rela ted channels reported here provides a structural rationale for the preferen ce of PC4(CTD) for juxtaposed single-stranded regions (e.g. in heteroduplex es) observed in earlier work.