Secondary structure of the pore‐forming colicin A and its C‐terminal fragment

Abstract

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A ( M , 60000), and a C-terminal bromelain fragment ( M , 20000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly a-helical with an enrichment of the a-helical content in the C-terminal domain carrying the ionophoric activity. The nonnegligible fl-sheet structure in the C-terminal domain is unstable and is easily transformed into a-helix upon decreasing the polarity of the solvent. N o evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane x-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic moment calculations. Similar calculations on the C-terminal domains of colicin E l and IB indicate a common structure of the pores formed by colicin A, E l and I,,. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, Is or E l . As a result, it is concluded that the channel is probably not built by ;I single colicin molecule but more likely by an oligomer.Colicins are bacteriocidal proteins ( M , 60000 -80000) which kill sensitive Eschevichia coli cells upon binding to a specific receptor located on the outer membrane of the cell, followed by translocation across the membrane(s) and biochemical interactions with their target in the cell. At present, there is a great interest in the colicins, such as colicin E l , A, K and IHr which interfere with membrane energization (for reviews see [I, 21).These soluble protein increase the conductivity of phospholipid bilayers by inducing the formation of voltagedependent channels. The voltage dependence of the channels formed by colicin A is strongly reminiscent of that observed for excitable membranes [3 -61. These colicins are suitable models relevant for understanding the molecular mechanism of channel formation and voltage gating of pores.The sequences of colicins E l , A and IB have been deduced from the nucleotide sequence of the corresponding genes [7, 81 (and Mankovich and Koninsky, personal communication [9-141.characteristics. Indeed, the size of the pore, as well as its voltage dependence, is modified upon protonation of an amino acid residue(s), the pK of which is 5.8. As to the conformations involved in pore formation, although some membrane proteins such as matrix porin of E. coli outer membrane have been shown to adopt a P-sheet structure, there is much evidence that protein segments interacting with lipids are a-helical. Amphipathic a-helices were proposed for serum apolipoproteins [IS], inelittin [19], calcitonin [20] and hormones or synthetic ana...

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins

“…The CD spectrum of the peptide ([10 in Fig. 7) was almost identical to that previously reported (Pattus et al, 1985;Lakey et al, 1991), and the decomposition results of the spectrum resulted in an a-helical content higher than 70'70, with less than 10% of strand, which coincided with the prediction result.…”

A 136‐amino‐acid‐residue COOH‐terminal fragment of colicin A is endowed with ionophoric activity

“…This would mean that helices 1 and 2, recently identified in the three-dimensional structure of colicin A [4], are not required for channel formation. It has been reported that the a-helical content of the lipid-associated COOH-terminal fragment of colicin A is similar to that of the soluble form [5]. This suggests that about the same helices as that identified in the soluble form [4] exist in the membraneinserted form of this fragment.…”

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors

“…Moreover, the segment of uncharged amino acids between residues 448 and 496 of colicin B was exactly the same length (49 residues) as the equivalent region in colicin A. It has been shown for colicin A that this region contains an a-helical segment and is essential for channel formation in membranes (32). It is reasonable to assume that this domain of colicin B has a similar conformation and is responsible for the channel formation which has been shown previously (33).…”