Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA

Abstract

The ccd toxin-antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdB(Vfi)) was crystallized in two different crystal forms. The first form belongs to space group I23 or I2(1)3, with unit-cell parameter a = 84.5 A, and diffracts to 1.5 A resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 A, beta = 110.0 degrees, and diffracts to 1.7 A resolution. The complex of CcdB(Vfi) with the GyrA14(Vfi) fragment of V. fischeri gyrase crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 A, and diffracts to 2.2 A resolution. The corresponding mixed complex with E. coli GyrA14(Ec) crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 A, beta = 102.6 degrees, and diffracts to 1.95 A. Finally, a complex between CcdB(Vfi) and part of the F-plasmid antitoxin CcdA(F) crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 A, and diffracts to 1.9 A resolution.

Figure 1

Amino-acid sequence alignment of the CcdB proteins of the E. coli F plasmid and its V. fischeri chromosomal homologue and amino-acid sequence alignment of the GyrA14 fragments from E. coli and V. fischeri. Amino acids that are not conserved are indicated in red. The essential Trp99 of the F-plasmid CcdB and its equivalent Thr103 of the CcdB of V. fischeri are highlighted in yellow, as is the conserved Arg462 of the two gyrase fragments.

Figure 2

Crystal habits. All photographs are on the same scale, with a common scale bar shown in (a). (a) Crystal of CcdB_Vfi (condition 1). (b) Crystals of CcdB_Vfi (condition 2). (c) Crystals of the CcdB_Vfi–GyrA14_Ec complex. (d) Crystals of the CcdB_Vfi–GyrA14_Vfi complex. (e) Crystals of the CcdB_Vfi–CcdA_F37–72 complex.