Abstract:
Colicin Ia is a 210 Å long, 69 kDa soluble protein that parasitises the outer membrane of sensitive Escherichia coli cells to form non-selective voltage gated channels. The molecular structure and organisation of its three domains namely: the receptor domain (R), the translocator domain (T) and the channel forming domain (C) are well understood from X-ray crystallographic studies. Based on numerous electrophysiological studies, it is known that monomeric colicin Ia binds to and co-opts its receptor (Cir) at outer membrane to translocate across the periplasmic space and form voltage gated ion channels in the plasma membrane. The formation of channel destabilises the cell membranes, causes efflux of ions and ultimately leading to cell death. In order, to do this the colicin Ia monomer has to undergo significant conformational changes i.e from a soluble state to a membrane inserted state exposing its hydrophobic residues in the lipid bilayer. Earlier studies had indicated the channel to be formed by a monomer. This notion conflicts with theoretical modelling which shows a shortfall of 20 amino acid residues needed to span the periplasmic space. On the other hand, visualisation at modest resolution by electron microscopy showed the putative channel to be formed by an oligomer associated with bilayer. In the current study, to further strengthen our understanding of alteration in molecular structure en route to channel formation, non-denaturing concentrations of urea was used. This resulted in formation of putative colicin Ia channel like “rings” which were strikingly similar to colicin Ia oligomer observed earlier. Furthermore, during lipid/detergent mediated 2D crystallisation and nanodisc experiments a number of colicin Ia rings were observed that remained soluble and not associated with lipid bilayer/sheets. Taken together, these results indicate that interaction with lipids promotes oligomerisation of colicin Ia but there might be other contributing factors that lead to membrane insertion. Ability to produce putative membrane inserted colicin Ia oligomers in soluble form as shown in this study, could pave the way for obtaining high resolution structural details of colicin Ia oligomer by X-ray crystallography.