Orientational Distribution of α-Helices in the Colicin B and E1 Channel Domains:  A One and Two Dimensional ¹⁵N Solid-State NMR Investigation in Uniaxially Aligned Phospholipid Bilayers

Abstract

Thermolytic fragments of the channel-forming bacterial toxins colicin B and colicin E1 were uniformly labeled with the 15N isotope and reconstituted into uniaxially oriented membranes. These well-aligned samples were investigated by proton-decoupled 15N solid-state NMR spectroscopy at 40.5 and 71.0 MHz. The one dimensional spectra indicate a predominant orientation of the colicin alpha-helices parallel to the bilayer surface but also the presence of a considerable proportion of peptide bonds that align in a transmembrane direction. The orientational distribution of 15N-labeled amide bonds is nearly identical for colicin B and E1, each a representative of a different group of membrane-active colicins. This comparison indicates common structural features of the water-soluble as well as the bilayer-associated proteins. When the pH is lowered, the orientational distribution of amide vectors exhibits only a small shift from in-plane to transmembrane orientations, in agreement with increased affinity and activity of colicins at acidic conditions. The 15N spectral line shape was independent of the bilayer phospholipid composition (100-75 mol % phosphatidylcholine/0-25 mol % phosphatidylglycerol) or the protein-to-lipid ratio in the range 1.7 - 12 wt %. Two dimensional separated local field spectroscopy (PISEMA) resolves almost 200 15N resonances of the colicin B channel protein. Approximately 50 15N signals resonate in a region characteristic of transmembrane helical residues, in strong support of the previously suggested umbrella conformation of the closed colicin channel.

Calorimetric Investigations of the Structural Stability and Interactions of Colicin B Domains in Aqueous Solution and in the Presence of Phospholipid Bilayers

“…7 and 8). This is in excellent agreement with previous structural findings for the colicin A pore-forming domain (19,25) as well as the high degree of primary sequence homology between the pore-forming fragments of ColA and ColB (16). The calorimetric, fluorescence, and circular dichroism studies presented in this paper demonstrate that the two reported transitions of ColB are due to denaturation of two discrete domains of the protein.…”

“…The full-length protein as well as its thermolytic fragment were purified following procedures described elsewhere (15,16). This latter domain of the homologous colicin A protein has been shown to exhibit pore-forming activities (e.g.…”

“…Proton-decoupled 15 N solid-state NMR spectra of oriented membrane samples indicate that the helices of the ColB and ColE1 C-terminal domains are predominantly orientated parallel to the bilayer surface (16). In addition, neutron scattering experiments show a location of large parts of the ColA PFF close to and beyond the membrane surface (47).…”

“…Although the isolated C-terminal fragment by itself is competent to insert into membranes (16), and pore-forming domains of other colicins exhibit pore-forming activities in vitro (17,(43)(44)(45), all three domains are required for recognition, translocation, and pore formation at intact cells in vivo. It appears that mutual interactions between different regions of colicin B help to maintain the translocation and the poreforming domains in less stable but functionally competent conformations.…”

The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner

“…Structurally, Bcl-x L ΔTM is most similar to the translocation domain of diphtheria toxin (23). For this toxin, as well as others, membrane association is mediated in part by the so-called hydrophobic, helical hairpin, which corresponds to α-helices 5 and 6 in Bcl-x L ΔTM (Figure 5a) (38)(39)(40)(41)(42)(43)(44)(45)(46). Despite the structural similarity, the sequences of these proteins are less than 19% identical, and they possibly are an example of convergent evolution toward a versatile protein fold.…”