The interface of a membrane-spanning leucine zipper mapped by asparagine-scanning mutagenesis

Abstract

An oligo-leucine sequence has previously been shown to function as an artificial transmembrane segment that efficiently self-assembles in membranes and in detergent solution. Here, a novel technique, asparagine-scanning mutagenesis, was applied to probe the interface of the self-assembled oligo-leucine domain. This novel approach identifies interfacial residues whose exchange to asparagine leads to enhanced self-interaction of transmembrane helices by interhelical hydrogen bond formation. As analyzed by the ToxR system in membranes, the interface formed by the oligo-leucine domain is based on a leucine-zipper-like heptad repeat pattern of amino acids. In general, the strongest impacts on self-assembly were seen with asparagines located around the center of the sequence, indicating that interaction is be more efficient here than at the termini of the transmembrane domains.

Figure 1.

Mapping interfacial residue positions of the oligo-leucine TMS (leu20) by asparagine-scanning mutagenesis in membranes. (A) Self-assembly of leu20 and its asparagine mutants as determined by the ToxR system. Mutants with strongly increased β-galactosidase activities (mean ± SE, n = 12) relative to the parental leu20 sequence follow a heptad repeat pattern and are labeled accordingly. (B) Protein expression levels were comparable as confirmed by Western blot. The order of samples corresponds to that in A. (C) Growth kinetics of PD28 cells expressing the ToxR constructs in minimal medium with maltose were similar, thus indicating similar concentrations of the different ToxR proteins in the membrane. A construct in which the TMS had been deleted (ΔTM) served as negative control. (D) Alignment of leu20 to the heptad pattern revealed by asparagine-scanning mutagenesis. The helical wheel diagram depicts how a and d positions form the interface of a leucine zipper. Although a pair of helices is shown for the sake of simplicity, the same geometry of side-chain packing applies to complexes with more than two helices.

Figure 2.

Self-assembly in detergent solution as analyzed by SDS-PAGE analysis. Proteins overexpressed in TOP10 cells were solubilized, electrophoresed at equivalent starting concentrations in sample buffer, and visualized by Western blotting. (Left) Electrophoresis of the leu20 protein not boiled in SDS sample buffer. (Right) leu20 was monomeric upon sample boiling, whereas its asparagine mutants still formed various oligomeric species when asparagine was located between positions 8 and 15.