Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae

Pilus fibers are long protein filaments on many pathogenic bacteria that participate in attachment to host cells. Although the self-assembling protein pilin is the major structural component of the Neisseria gonorrhoeae pilus fiber, several other proteins co-purified with pilin through the repeated solubilization-reassociation steps of the biochemical purification. Pilin solubilized in the nondenaturing detergent n-octyl-β-D-glucopyranoside remained an aggregate of about 100 kDa at pH 9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly involves electrostatic interactions of lysine, tyrosine, or other side chains with high pK_a values. Pilin dimers and aggregates of higher molecular mass were partially stable even in the presence of sodium dodecyl sulfate and β-mercaptoethanol. Removal of pilus-associated proteins and stabilization of pilin multimers permitted the re producible crystallization of pilin. Three-dimensional needle- and plate-shaped crystals of purified N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40% polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell dimensions a= 126.4, b= 121.2, c = 26.7 Å and V_m = 2.84 ų/dalton for one molecule per asymmetric unit. The best crystals diffracted to 2.4 Å resolution using synchrotron radiation, were stable to x-ray damage, and appear suitable for determination of the atomic structure. This approach of stabilizing and crystallizing an intermediate assembly state may be useful for other fiber-forming proteins, which have previously not been successfully crystallized in forms that diffract to atomic resolution.