Abstract
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 P3(1)21 or P3(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.
Highlights
Bacterial surfaces are decorated by sticky hair-like structures called fimbriae or pili that allow them to recognize abiotic surfaces, host receptors and each other (Kline et al, 2009; Proft & Baker, 2009)
Different strains of E. coli have developed specific pili to enable them to thrive in their niche environments, almost all produce a surface fibre called the E. coli common pilus (Ecp; Pouttu et al, 2001; Rendon et al, 2007; Garnett et al, 2012)
We describe our preliminary crystallographic analyses of EcpB and envisage that the elucidation of its structure will further unravel the anomalies in this alternative CU pathway
Summary
Bacterial surfaces are decorated by sticky hair-like structures called fimbriae or pili that allow them to recognize abiotic surfaces, host receptors and each other (Kline et al, 2009; Proft & Baker, 2009). Different strains of E. coli have developed specific pili to enable them to thrive in their niche environments, almost all produce a surface fibre called the E. coli common pilus (Ecp; Pouttu et al, 2001; Rendon et al, 2007; Garnett et al, 2012) This structure is involved in key processes during sessile Enterobacteriaceae lifecycles, where it mediates both host-cell adherence and early biofilm interbacterial interactions (Rendon et al, 2007; Lehti et al, 2010; Garnett et al, 2012). EcpD is an adhesive-tip subunit that can recognize receptors on the surface of host cells It is the largest pilin subunit of all known CU systems (57.7 kDa) and has the unique ability to self-polymerize (Garnett et al, 2012; Rossez et al, 2014). We describe our preliminary crystallographic analyses of EcpB and envisage that the elucidation of its structure will further unravel the anomalies in this alternative CU pathway
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