Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins.

The interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein-protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1-α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1-α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein-protein interactions, with implications for drug development and rational engineering of these interfaces.

Identification of channel-lining amino acid residues in the hydrophobic segment of colicin Ia.

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of approximately 177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 alpha-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573-616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as alpha-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.

The effect of protein complexation on the mechanical stability of Im9.

Force mode microscopy can be used to examine the effect of mechanical manipulation on the noncovalent interactions that stabilize proteins and their complexes. Here we describe the effect of complexation by the high affinity protein ligand E9 on the mechanical resistance of the simple four-helical protein, Im9. When concatenated into a construct of alternating I27 domains, Im9 unfolded below the thermal noise limit of the instrument ( approximately 20 pN). Complexation of E9 had little effect on the mechanical resistance of Im9 (unfolding force approximately 30 pN) despite the high avidity of this complex (K(d) approximately 10 fM).

On the role of lipid in colicin pore formation.

Insights into the protein-membrane interactions by which the C-terminal pore-forming domain of colicins inserts into membranes and forms voltage-gated channels, and the nature of the colicin channel, are provided by data on: (i) the flexible helix-elongated state of the colicin pore-forming domain in the fluid anionic membrane interfacial layer, the optimum anionic surface charge for channel formation, and voltage-gated translocation of charged regions of the colicin domain across the membrane; (ii) structure-function data on the voltage-gated K(+) channel showing translocation of an arginine-rich helical segment through the membrane; (iii) toroidal channels formed by small peptides that involve local participation of anionic lipids in an inverted phase. It is proposed that translocation of the colicin across the membrane occurs through minimization of the Born charging energy for translocation of positively charged basic residues across the lipid bilayer by neutralization with anionic lipid head groups. The resulting pore structure may consist of somewhat short, ca. 16 residues, trans-membrane helices, in a locally thinned membrane, together with surface elements of inverted phase lipid micelles.

Mapping the membrane topology of the closed state of the colicin E1 channel.

The membrane-associated closed channel state of the colicin E1 thermolytic peptide was studied by the Parallax Method of depth-dependent fluorescence quenching. A number of single Trp-containing peptides of colicin E1 were prepared to facilitate the use of Trp as a probe for the topography of the channel peptide in the membrane-bound state. The bound form of the channel peptide was studied by binding channel peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-pal-mitoyl -2-oleoyl-sn- glycero-3-phosphatidylglycerol large unilamellar vesicles (60:40, mol/mol, approximately 0.1 microns diameter vesicles prepared by an extrusion technique) at low pH (pH 3.5). Depth-dependent fluorescence quenching studies using two nitroxide-labeled phospho-lipids (1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphatidylcho++ +-line and 1-palmitoyl-2-(12-doxylstearoyl)-sn-glycero-3-phosphatidylcholine) were conducted to determine the membrane location of each Trp residue for the vesicle-bound peptide. The three naturally occurring Trp residues in the colicin channel peptide, Trp-424, Trp-460, and Trp-495, were found to reside at membrane depths (from the C-2 carbon of the fatty acyl chain) of 7.4, 3.1, and 8.4 A, respectively. Three Trp residues (Trp-355, Trp-460, and Trp-507) in the channel peptide were classified as shallow (0-5.0 A from C-2 carbon). The remaining 9 Trp residues were classified as moderately buried (5.1-10.0 A). None of the dozen tryptophyls were classified as deeply buried in the membrane bilayer (10.1-15.0 A). A model for the colicin E1 channel based on these measurements along with previous data obtained from proteolysis, chemical labeling, ESR quenching, and mutagenesis experiments is proposed. This model for the closed state of the channel has as its central feature of the presence of only two trans-membrane segments. The membrane-associated portion of the channel includes the hydrophobic membrane anchor domain, Ala-474 to Ile-508. Furthermore, the fluorescence quenching data are consistent with the NH2-terminal helices (helices 1-7) lying on the surface of the membrane with the helical axis being oriented parallel to the membrane plane.

Membrane-bound form of the pore-forming domain of colicin A. A neutron scattering study.

The ion-channel forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. This fragment forms a well-defined complex with dimyristoylphosphatidyl-glycerol which is thus amenable to neutron scattering studies. Neutron scattering experiments in the Guinier range (low angles) provided the mass and the stoichiometry of the complex (290,000 (+/- 10,000) M(r), 8.2 (+/- 0.5)), in fair agreement with previous determinations. By varying the neutron scattering length density of the solvent with 2H2O/H2O mixtures and therefore the contrast of the different components, the radial distribution of the protein and of the lipids was determined. Finally, an attempt was made to fit various models to the wider angle scattering data. This study suggests that the pore-forming fragment of colicin A lies mostly at the surface of the membrane, with the lipids arranged in a bilayer organization.