Crystallization and X-ray diffraction analysis of the complement component-3 (C3) inhibitory domain of Efb from Staphylococcus aureus

Abstract

The extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus is a multifunctional virulence factor capable of potent inhibition of complement component-3 (C3) activity in addition to its previously described fibrinogen-binding properties. A truncated recombinant form of Efb (Efb-C) that binds C3 has been overexpressed and purified and has been crystallized using the hanging-drop vapor-diffusion technique. Crystals of native Efb-C grew in the tetragonal space group P4(3) (unit-cell parameters a = b = 59.53, c = 46.63 A) with two molecules in the asymmetric unit and diffracted well beyond 1.25 A limiting Bragg spacing. To facilitate de novo phasing of the Efb-C crystals, two independent site-directed mutants were engineered in which either residue Ile112 or Val140 was replaced with methionine and crystals isomorphous to those of native Efb-C were reproduced using a seleno-L-methionine-labeled form of each mutant protein. Multiwavelength anomalous diffraction (MAD) data were collected on both mutants and analyzed for their phasing power toward solution and refinement of a high-resolution Efb-C crystal structure.

Figure 1

Representative sections of anomalous Patterson maps calculated from selenium MAD data of either the I112M (a) or V140M (b) mutant of Efb-C. 2.2 Å limiting resolution maps are shown in fractional coordinates at section z = 0.263 with a contour level of 0.5σ starting at 2σ. In both cases, the number of visible peaks is consistent with three crystallographically unique selenium sites according to the N(N − 1) formalism.