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

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Michal HammelBrian V Geisbrecht

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.

References

Jan 1, 1994·Annual Review of Microbiology·J M PattiM Höök
Feb 5, 1999·Clinical and Experimental Immunology·E SakinieneA Tarkowski
Mar 25, 1999·Acta Crystallographica. Section D, Biological Crystallography·T C Terwilliger, J Berendzen
Jun 18, 2003·The Journal of Biological Chemistry·Brian V GeisbrechtDaniel J Leahy
Jul 10, 2004·The Journal of Infectious Diseases·Lawrence Y L LeeEric L Brown
Feb 5, 2005·The Journal of Biological Chemistry·Brian V GeisbrechtDaniel J Leahy
Nov 2, 2005·Protein Expression and Purification·Brian V GeisbrechtMihai Pop

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Citations

Mar 14, 2007·Nature Immunology·Michal HammelBrian V Geisbrecht
Aug 19, 2007·The Journal of Biological Chemistry·Michal HammelBrian V Geisbrecht

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