Cloning, purification, crystallization and preliminary X-ray diffraction studies ofEscherichia coliPapD-like protein (EcpD)

Abstract

Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å . There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å 3 Da À1 , with 59% solvent content. Initial phases were determined by molecular replacement.

Crystal structure of the usher chaperone YadV reveals a monomer with the proline lock in closed conformation suggestive of an intermediate state

“…Thus, the self-capping mechanism has been hypothesized to explain the stoichiometry of chaperone and pilus subunit. Our findings from Stokes radius calculation (2.23 nm) from gel permeation chromatography suggest that YadV is present as a monomeric form in solution as high as~1 mM concentration of protein [30]. In agreement with these solution state data, the crystal structure has one monomer of YadV in the asymmetric unit.…”

“…Cloning, overexpression, and purification details are described in our previous study [30]. Briefly, YadV was cloned in pET11a vector without any affinity chromatography purification tag and periplasmic signal peptide.…”

“…Gel permeation chromatography was carried out to calculate the Stokes radius (R s ) of YadV as reported in our previous study [30]. In brief, the gel phase distribution coefficient…”

“…The detailed crystallization process can be found in our previous publication [30]. Briefly, the diffraction quality YadV crystals were obtained in hanging drop vapor diffusion method in a solution of 0.1 M ammonium sulfate, 0.1 M sodium acetate pH 4.6, 0.3 M sodium formate, and 30% (w/ v) PEG4000 at 277 K. A single crystal was flash-frozen under a stream of nitrogen gas at 100 K. The crystal was diffracted up to 1.63 A resolution, and unit cell parameters were a = 100.3; b = 127.6; and c = 45.9…”