Design of a heterotetrameric coiled coil

Abstract

We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401-1407). We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.

Figure 1

Backbone representation of the coiled coil highlighting single glu-lys salt-bridges between individual chains. The sidechains were energy minimized as described in the Materials and Methods and the distances between the carboxy and amino groups after energy minimization are highlighted. The peptide sequences are shown for the parent peptide GCN4-pLI, ecE, and ecK, with substitutions color-highlighted.

Figure 2

Circular dichroism spectra of ecE and ecK peptides were collected either individually (dashed and dashed/dotted lines) or as a 1:1 mixture (solid line). Samples were prepared in 10 mM phosphate, pH 7.5, and 150 mM NaCl at 25°C.

Figure 3

Determination of oligomeric state by analytical ultracentrifugation. (A) Sedimentation velocity experiments were prepared using 400 μM peptide monomer concentration in 10 mM phosphate, pH 7.3, 150 mM NaCl at 25°C. The data are shown fit with a single species with an s_20,w = 1.400 ± 0.001 S and a D_20,w = 11.81 ± 0.03 × 10⁻⁷ cm² s⁻¹. The residuals from fitting of the model to the data are shown below. (B) Sedimentation equilibrium experiments were run at 30,000, 40,000, and 50,000 rpm. The solution conditions are identical to the SV experiment described in A) except that the peptide concentration was 30 μM (on a per monomer basis). The data at all three speeds are shown fit with a single species model in which the molecular mass was a fitting parameter. The residuals from fitting of the model to the data for each speed are shown below for 50,000 rpm (top), 40,000 rpm (middle), and 30,000 rpm (bottom).

Figure 4

Fluorescence analysis of 1-anilinonaphthalene-8-sulfonate (ANS) binding. Protein samples were measured in 10 mM sodium phosphate, pH 7.5, and 150 mM NaCl. All samples contained 1 μM ANS. Samples: 90% ethanol (black); 6 μM apomyoglobin (green); 6 μM lysozyme (red); 6 μM ecE/ecK (blue).

Figure 5

pH dependence on folding of 3 μM ecE and ecK separately measured by CD signal at 222 nm, in 1 mM each of sodium phosphate, sodium borate, and sodium citrate and 150 mM NaCl.

Figure 6

Denaturation profile of ecE/ecK mixture in 10 mM sodium phosphate, pH 7.5 with and without 150 mM NaCl at 25°C as monitored by circular dichroism.