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Purification, crystallization and preliminary X‐ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB
Author(s) -
Garnett James A.,
Diallo Mamou,
Matthews Steve J.
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x15006354
Subject(s) - periplasmic space , chaperone (clinical) , pilus , escherichia coli , biogenesis , crystallography , crystallization , fimbria , chemistry , biophysics , biofilm , fimbriae proteins , bacteria , biology , biochemistry , gene , medicine , genetics , organic chemistry , pathology
Pili are key cell‐surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli , the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host‐cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting‐drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P 3 1 21 or P 3 2 21, with unit‐cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine‐substituted protein and heavy‐atom derivatives are being prepared for phasing. The three‐dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.