Identifying ionic interactions within a membrane using BLaTM, a genetic tool to measure homo- and heterotypic transmembrane helix-helix interactions

Abstract

The assembly of integral membrane protein complexes is frequently supported by transmembrane domain (TMD) interactions. Here, we present the BLaTM assay that measures homotypic as well as heterotypic TMD-TMD interactions in a bacterial membrane. The system is based on complementation of β-lactamase fragments genetically fused to interacting TMDs, which confers ampicillin resistance to expressing cells. We validated BLaTM by showing that the assay faithfully reports known sequence-specific interactions of both types. In a practical application, we used BLaTM to screen a focussed combinatorial library for heterotypic interactions driven by electrostatic forces. The results reveal novel patterns of ionizable amino acids within the isolated TMD pairs. Those patterns indicate that formation of heterotypic TMD pairs is most efficiently supported by closely spaced ionizable residues of opposite charge. In addition, TMD heteromerization can apparently be driven by hydrogen bonding between basic or between acidic residues.

Figure 1. Design and validation of the BLaTM assay.

(a) Scheme depicting the main features of the system. (b) Example of dose-response curves where the decrease of E. coli cell density (A₅₄₄) to increasing ampicillin concentration is used to calculate LD₅₀ values. (c) LD₅₀ values derived from part. (b) These values characterize the homotypic interaction of the wild-type (wt) GpA and QSOX2 TMDs and the impact of point mutations (mut). TMDs were inserted into the hybrid proteins at orientations where wild-type/mutant LD₅₀ ratios were maximal (see: Supplementary Fig. S4). (d) Sequence-specific homo- and heterotypic interaction driven by ionizable residues. The LS46 orientation used here leads to superior LD₅₀ values compared to other orientations (not shown). All data were generated using BLaTM 1.1 in E. coli BL21 (b,c) or in E. coli JM83 cells (d) and represent means ± SEM, n = 4 separate transformations. The denomination D₆R₇ corresponds to D₅R₆ previously used. Single, double, or triple asterisks denote statistical significance at the 0.05, 0.01, or 0.001 confidence levels (relative to the mutants in part (c) or to L19GG in (d)).

Figure 2. Influence of expression level on ampicillin resistance and wild-type/mutant discrimination.

(a) GpA wild-type and mutant TMD frames tested. (b) Effect of IPTG concentration on ampicillin resistance. Lowering the activity of the pBAD promoter by increasing the IPTG concentration (133 μM arabinose) influences LD₅₀ in a way that depends on TMD orientation. No data points are given for GpA₊₁ wt at 0.1 mM and 0.2 mM IPTG since the corresponding LD₅₀ values were too high for reliable determination. (c) For control, protein expression was analyzed by GFP fluorescence that was normalized to cell density (A₅₄₄, A.U. = arbitrary units). Even if the GFP moiety is proteolytically cleaved off from part of the BLa proteins during expression (see: Supplementary Fig. S4d), GFP fluorescence represents the amount of originally expressed protein. Note that the expression level varies as a function of induction but not of TMD orientation or sequence. All TMDs are expressed in BLaTM 1.2 in E. coli JM83 where the expression level is too low for detection by Western blotting. Means ± SEM, n = 3.

Figure 3. List of isolated TMD pairs sorted by affinity.

¹The sequence above the identified sequence pairs represents the randomized model based on L19GG (X = R, K, E, or D). Dots in the identified sequences represent Leu. ²Single letter code of isolated N-BLa (upper sequence)/C-BLa (lower sequence) combination. ³In % (GpA₊₁ wt = 100%; GpA₊₁ G83I = 29 ± 3%; L19GG = 37 ± 4%); means ± SEM, n = 4–12. ⁴Number of times the sequence pair was found in the selected TMDs.

Figure 4. Design of combinatorial library and analysis of TMD pairs.

(a) Pattern of randomized positions within the L19GG background. Each sequence contains a single Glu, Asp, Lys, or Arg at one of the positions X. Flanking sequences (lower case) represent the NheI/BamHI restriction sites. (b–d) An evaluation of the TMD pairs that were isolated by growth on ampicillin and sorted into high, medium and low affinity based on their individually measured LD₅₀ values (Fig. 3). Left panels: Occurrence of pairs with oppositely charged (+/−, −/+; black bars) or like charged (+/+, −/−; grey bars) residues as well as pairs where only one TMD contained an ionizable residue (light grey). Right panels: Distribution of ionizable residues along the isolated TMD sequences encoded by N-BLa (upward pointing bars) or C-BLa (downward pointing bars) plasmids. Note that high-affinity TMD pairs tend to have oppositely charged residues mainly at positions 3, 6, and 7 and that positively and negatively charged amino acids are found in TMDs encoded by N-BLa as well as by C-BLa plasmids.

Figure 5. Selection of TMD pairs from a combinatorial library.

(a) LD₅₀ values were determined using BLaTM 1.2 in E. coli JM83 cells and are shown relative to wild-type GpA₊₁ (=100%) as listed in Fig. 3. Protein expression was induced with 133 μM arabinose plus 0.3 mM IPTG. Horizontal broken lines separate low-affinity pairs (<50% GpA) from medium-affinity (50% to 80% GpA) and high-affinity (>80% GpA) pairs. (b) For control, protein expression levels were analyzed by GFP fluorescence normalized to cell density and wild-type GpA₊₁ (=100%). Note that the expression level does not increase with increasing LD₅₀; therefore, higher ampicillin resistance is caused by specific TMD interactions, rather than by increased protein expression. Means ± SEM, n = 4–12.