Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant

Abstract

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.

Figure 1.

Crystal structure of the GCN4-pAe tetramer. (A) Lateral view of the tetramer. The sequence of the GCN4-pAe peptide is MK VKQLADK VEELLSK NYHLANE VARLAKL VGER, with e heptad positions underlined. Residues at the a positions are shown as red van der Waals surfaces, and the d positions are green. Blue van der Waals surfaces identify Ala21(e) and Ala28(e), and pink surfaces identify Glu23(g) and Leu30(g). The N termini of the A–B (cyan) and C–D (light pink) dimers are indicated. (B) Cross-section of the A–B dimer in the Leu13(d) layer. The 1.40 Å 2F _obs – F _calc electron density map (contoured at 1.5σ) is shown with the refined molecular model. (C) Cross-section of the A–B dimer in the Asn17(a) layer. Water molecules are shown as red spheres. Hydrogen bonds are denoted by pink dotted lines, and hydrogen-bond distances are given in Å. (D) Superposition of the backbone conformations of the GCN4-pAe tetramer and two wild-type leucine-zipper dimers (green).

Figure 2.

Interfacial interactions between GCN4-pAe dimers. (A) Side view showing side-to-side aggregation of dimers. The side chains of residues 19–32 that form the dimer–dimer interface are displayed. (B) Helix-to-helix packing in the Leu-A20(d)–Leu-C30(g)–Val-D31(a) layer. The 1.40 Å 2F _obs – F _calc electron density map contoured at 1.5σ is shown with the refined molecular model. (C) Helix to helix packing in the Val-B24(a)–Leu-C27(d)–Ala-D28(e) layer. (D) Helical wheel projection of residues 19–32 of the GCN4-pAe tetramer, depicting interdimer packing interactions (dotted lines).