A seven-helix coiled coil

Abstract

Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between alpha-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable alpha-helical heptamer in aqueous solution. The 1.25-A resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of alpha-helices.

Fig. 1.

Helical wheel projection of residues Met-1 to Arg-34 of the GCN4-pAA sequence. The view is from the N terminus. Heptad repeat positions are labeled a through g. GCN4-pAA differs from the recombinant dimeric leucine-zipper peptide GCN4-pR by alanine substitutions at four e and four g positions (bold). The sequences of GCN4-pR and GCN4-pAA (with the eight mutated e and g positions set in italics) are as follows: GCN4-pR, MK VKQLEDK VEELLSK NYHLENE VARLKKL VGER; GCN4-pAA, MK VKQLADA VEELASA NYHLANA VARLAKA VGER. The GCN4-pR sequence contains an additional Met-Lys-Val and no Arg-Met at its N terminus but is otherwise identical to GCN4-p1.

Fig. 2.

The GCN4-pAA peptide forms a seven-stranded helical bundle. (A) CD spectrum at 20°C in ABS (pH 5.2) and 50 μM peptide concentration. (B) Thermal melt monitored by CD at 222 nm. (C) Representative sedimentation equilibrium data of a 100 μM sample at 20°C and 18,000 rpm in ABS (pH 5.2). The data fit closely to a heptameric complex. (Upper) The deviation in the data from the linear fit for a heptameric model is plotted.

Fig. 3.

Crystal structure of GCN4-pAA. (A) Lateral view of the heptamer (residues 5–31). Red van der Waals surfaces identify residues at the a positions, and green surfaces identify residues at the d positions. (B) Axial view of the heptamer. The view is from the N terminus looking down the superhelical axis. Red spheres identify residues at the a positions, green spheres identify residues at the d positions, yellow spheres identify residues at the e positions, and pink spheres identify residues at the g positions. (C) Molecular surface representation of the heptamer. The solvent-accessible surface is colored according to the local electrostatic potential, ranging from +32 V in dark blue (most positive) to −30 V in deep red (most negative). (D) Cross-section of the superhelix in the Asn-17(a) layer. The 1.25-Å 2F_o − F_c electron density map at 1.5σ contour is shown with the refined molecular model. Hydrogen bonds are denoted by pink dotted lines. (E) Omit map showing the Leu-20(d) layer in a 2F_o − F_c difference Fourier synthesis (contoured at 1.5σ). (F) Helical wheel projection of the heptamer showing interhelical packing interactions. The view is from the N terminus. Heptad positions are labeled a through g.

Fig. 4.

Comparison of helix–helix interfaces in parallel coiled coils. (A) Parallel packing at position a (180°) in the GCN4-p1 dimer (23). The view is from the N terminus looking down the superhelical axis. The C^α–C^β bond of each knob (blue) is oriented parallel to the C^α–C^α vector (red) at the base of the recipient hole on the neighboring helix. The C^α–C^β bonds of the e and g residues are shown in pink and cyan, respectively. (B) Acute packing at position a (120°) in the GCN4-pII trimer (52). (C) Perpendicular packing at position a (90°) in the GCN4-pLI tetramer (23). (D) Packing at position a (72°) in the Trp-14 pentamer (31). (E) Packing at position a (51.4°) in the GCN4-pAA heptamer. (F) Perpendicular packing at position d (90°) in the dimer. (G) Acute packing at position d (150°) in the trimer. (H) Parallel packing at position d (180°) in the tetramer. (I) Packing at position d (198°) in the pentamer. (J) Packing at position d (218.6°) in the heptamer.