Abstract
Periplasmic membrane-fusion proteins (MFPs) are an essential component of multidrug and metal-efflux pumps in Gram-negative bacteria. However, the functional structure of MFPs remains unclear. CusCFBA, the Cu(I) and Ag(I) efflux system in Escherichia coli, consists of the MFP CusB, the OMF CusC and the RND-type transporter CusA. The MFP CusB bridges the inner membrane RND-type efflux transporter CusA and the outer membrane factor CusC and exhibits substrate-linked conformational changes which distinguish it from other MFP-family members. CusB from E. coli was overexpressed and the recombinant protein was purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified CusB protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 3.1 A at 100 K. The crystal belonged to space group C222.