Pseudomonas aeruginosa uses long, thin fibres called type IV pili (T4P) for adherence to surfaces, biofilm formation, and twitching motility. A conserved subcomplex of PilMNOP is required for extension and retraction of T4P. To better understand its function, we attempted to co-crystallize the soluble periplasmic portions of PilNOP, using reductive surface methylation to promote crystal formation. Only PilOΔ109 crystallized; its structure was determined to 1.7 Å resolution using molecular replacement. This new structure revealed two novel features: a shorter N-terminal α1-helix followed by a longer unstructured loop, and a discontinuous β-strand in the second αββ motif, mirroring that in the first motif. PISA analysis identified a potential dimer interface with striking similarity to that of the PilO homolog EpsM from the Vibrio cholerae type II secretion system. We identified highly conserved residues within predicted unstructured regions in PilO proteins from various Pseudomonads and performed site-directed mutagenesis to assess their role in T4P function. R169D and I170A substitutions decreased surface piliation and twitching motility without disrupting PilO homodimer formation. These residues could form important protein-protein interactions with PilN or PilP. This work furthers our understanding of residues critical for T4aP function.