Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap pili is controlled by a phasevariation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. Methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap pili gene expression in both phase-ON and phase-OFF cells. We used modern nuclear magnetic resonance techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold closely resembles that of the winged helix-turn-helix family of DNA-binding proteins. We also determined that PapI possesses its own DNA-binding activity, albeit non-sequence specific, independent of Lrp. PapI appears to bind to DNA with a K d in the 10 µM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings.