Figure 5.

Crystal structure of rBcpA*(D³¹²A). (A) The crystal structure of rBcpA*(D³¹²A) was solved using molecular replacement. The CNA₂, XNA, and CNA₃ domains are shown as blue ribbons, the structure of the CNA₁ domain is not included in rBcpA*(D³¹²A). The Lys and Asn residues forming the intramolecular isopeptide bonds in the CNA₂ and CNA₃ domains are shown as black sticks. The intramolecular isopeptide bond in the XNA domain is not formed. The CNA₂ and CNA₃ domains assume reverse Ig-like folds and the XNA domain a jellyroll fold. (B+C) Close up of the XNA domain of rBcpA*(D³¹²A) (blue) and rBcpA* (magenta), in which the A³¹² and the catalytic residue D³¹² are in black and the K²⁷³ and N³⁸³ residues in magenta and blue and presented as sticks, respectively. In rBcpA*(D³¹²A), the K²⁷³-N³⁸³ intramolecular isopeptide bond between the two penultimate β-strands is not formed, as the catalyst D³¹² was replaced with alanine. Formation of the XNA isopeptide bond of rBcpA* (blue sticks) is accompanied by the loss of ammonium. (D) Treatment of rBcpA* and rBcpA*(D³¹²A) with (+) and without (-) trypsin protease revealed that the absence of the XNA isopeptide bond in rBcpA*(D³¹²A) renders the protein sensitive to protease; rBcpA* is protease resistant. (E) AFM images of wild-type (WT) and mutant D³¹²A pili showing the morphological difference between these two types of pili. (F) Thermal melt spectroscopy analysis as a function of temperature demonstrated that rBcpA*(D³¹²A) is less stable than rBcpA*.