Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Abstract

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

Keywords: OmpA; membrane proteins; protein design; statistical potential; β-barrel.

Fig. 1.

E_zβ was used to generate a membrane-oriented structure of E. coli OmpA from PDB 1bxw (46). Forty-two lipid-facing amino acids that faced the protein exterior, had >20% solvent-accessible surface area, and lay within a 25-Å membrane-spanning region were selected for computational redesign (highlighted in orange). Wild-type identities were maintained for lipid-facing positions 168 and 170 given their role in targeting of OmpA to the outer membrane. Interstrand backbone hydrogen bonds are shown as dotted lines. Extracellular and periplasmic regions are omitted for clarity.

Fig. S1.

Polyacrylamide gel electrophoresis analysis of designed OmpA expression. OmpA constructs contained a C-terminal FlAsH tag (CCPGCC). (Left) Total cell lysates; (Right) outer membrane fractions isolated by ultracentrifugation and solubilization with sarkosyl. (Upper) White light image showing all protein bands. (Lower) Fluorescence image showing only proteins that bound FlAsH. (Upper arrow) OmpA with periplasmic targeting sequence (191 amino acids, 20.7 kDa). (Lower arrow) OmpA without periplasmic targeting sequence (171 amino acids, 18.7 kDa). M = BenchMark protein ladder (Life Technologies), neg = negative control (WT without IPTG induction), WT = wild type, L = all-leucine design, L′ = L after restoration of Tyr168, 1–4 = OR1-4, 1′-4′ = OR1-4 after restoration of Tyr168. Although labeled bands are absent in the L and 1′ lanes, the high-molecular-weight background bands are also missing, suggesting poor yield from the periplasmic protein isolation.

Fig. S2.

Photographs of phage susceptibility plates. Two plates prepared on separate days are shown in separate panels. On each plate are spotted eight successive tenfold dilutions of K3 phage solution. Two identical sets were spotted on each plate.

Fig. 2.

OmpA membrane insertion was determined with two experimental methods. Data for wild-type OmpA, H2578, H1678, and OR4cons are shown. Data for all variants are provided as Supporting Information. (A) Phage susceptibility was tested by spotting successive tenfold dilutions of phage solution on a confluent plate of E. coli. Bacteriophage K3 infection depends upon the presence of properly folded OmpA. (B) Flow cytometry of E. coli cells induced with IPTG and incubated with FITC-labeled (indicated by a star) anti-HA antibodies. A fluorescent population indicates the presence of OmpA loops on the exterior of the cell.

Fig. S3.

Flow cytometry fluorescence histograms qualitatively agree with the phage assay results. OmpA variants were expressed in E. coli induced with IPTG. Binding of a fluorescently conjugated antibody against an affinity tag engineered into an external loop of OmpA produces a fluorescent signal in flow cytometry and indicates proper membrane insertion and folding. The nonfluorescent population in cases with two peaks is due to nonuniform IPTG uptake by E. coli cells.

Fig. S4.

Amino acid sequence alignment highlighting differences between wild-type OmpA, OR4, and OR4cons. The sequences of the eight β-strands are shown. Uppercase letters indicate redesigned positions. Vertical lines indicate mutations. WT: wild-type OmpA. OR4: OmpA redesign 4. OR4hybrid: the version of OR4 used as a parent in the creation of hybrids; some mutations at the ends of strands were reverted to facilitate hybrid assembly. OR4cons: conservative version of OR4 with additional mutational reversions.

Fig. 3.

Structural role of proline 121. P121 is unable to participate in a hydrogen bond with the backbone carbonyl of N145, ending the beta-strand hydrogen bond network between strands 6 and 7. Mutation to any other amino acid could propagate the strand further, altering the conformation locally. Other than for P121, only backbone atoms are depicted for clarity.

Fig. S5.

Insertion depth and sequence complexity constraints on design. (A) The depth-dependent insertion energy profile was calculated based on the E_zβ energy of lipid facing residues with the protein centered at a series of distances from the center of the lipid bilayer. OR4 (blue) is highly favored at the center of the bilayer with P = 0.8. Idealized shallow (red) and flat (green) insertion profiles have smaller values of P. Negative to positive depths correspond to insertion of the protein from the periplasmic to the extracellular direction. Profiles were computed as described previously (34). (B) The sequence complexity score increases with increasing amino acid diversity. Values are shown for a 43 residue sequence. Amino acid diversity corresponds to the number of amino acid types found in the sequence, where the number of amino acids are equally distributed as required to sum to 43 total residues (i.e., for diversity = 6, a potential sequence would be 7 Leu + 7 Val + 7 Ile + 7 Ala + 7 Phe + 8 Ser). The sequence complexity of wild-type OmpA and OR4 correspond to an amino acid diversity of nine.

Fig. S6.

Fractional factorial sampling of OR4 backcross hybrid variants to determine the strand contributions to folding. (A) The fractional factorial set consists of wild-type, OR4 (mut) and 14 backcross hybrids. Each strand is sampled as wild-type or mutant 8 times over the members of the set. The response observable (y) is the phage susceptibility score. (B) Strand effects (C_i) for strand i, computed as (∑_j (x_i,j × y_j))/16 where j ∈ (WT, mut, H1346 … H1248). (C) Half-normal plot of strand effects indicates strand 3 and strand 6 are significant factors. (D) strand pair interactions with strand 3 and strand 6 suggest significant pairwise contributions with strand 1 and/or strand 4.