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 ␣-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 ␣-helical heptamer in aqueous solution. The 1.25-Å 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 ␣-helices.protein design ͉ protein structure ͉ helix-helix interfaces ͉ buried polar interactions ͉ cavity H elix-helix interactions are ubiquitous in the native structure of proteins and in associations among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signaling and transport. Coiled coils afford a unique model system for elucidating principles of molecular recognition between helices (1-4). The conformation of coiled coils is prescribed by a characteristic 7-aa repeat, the 3-4 heptad repeat denoted as a-b-c-d-e-f-g (5). Positions a and d are typically occupied by hydrophobic amino acids such as Leu, Ile, Val, and Ala, whereas residues at positions e and g are frequently polar or charged (5-8). Crick's (9) classical analysis proposed that the nonpolar a and d side chains associate by means of complementary ''knobs-into-holes'' packing to form supercoiled ␣-helical ribbons. Beyond the role of hydrophobic side chains at the core a and d positions, coiled coils can use intra-and interhelical electrostatic interactions to tune their stability, especially those between the flanking e and g positions of neighboring chains (10-18). Despite their seeming regularity in sequence and packing patterns, coiled coils exhibit remarkable diversity in the number and arrangement of the associating helices: two-to five-stranded structures have been identified with parallel or antiparallel helix orientation (2). The structural parameters of dimers have been described by Crick (9), and general rules governing the close packing of ␣-helices in hexamers and dodecamers have been deduced (19,20).Analysis of coiled coils provides insight into the general problem of protein folding ...

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

“…In other words, the energy landscape for sequences based on a = Ile plus d = Leu is rugged with multiple minima that lie close in energy. This is consistent with literature reports of GCN4-based sequences, which have a = Val plus d = Leu and show a wide range of oligomer states, 13,[48][49][50] and with other design studies. 8,51,52 By contrast, CC-pII-I17N was markedly less folded and less thermally stable than any of the other peptides from either series, Table 1: it was only ≈50% helical at 10 µM concentration and 5˚C; and the TM was 36˚C, Figure S5B.…”

Crystal structure of a super leucine zipper, an extended two‐stranded super long coiled coil

“…For example, mutations of eight hydrophilic residues to hydrophobic alanine residues result in a seven-helix coiled coil. 15 These data seem to suggest a principle of folding and assembly of coiled coil that the more hydrophobic the helix, the higher oligomeric number the coiled coil. In the case of the leucine zipper mutant, the first three mutations of K3A, D7A, Y17W, and H18N result in more hydrophobic pimples or more hydrophobic surface area on the helix.…”

“…9 A series of leucine zipper mutants, which result from mutations of the residues on the interface between the two helices of the leucine zipper, have been designed to understand the effects of these mutations on coiled coil folding and assembly, such as the oligomeric state and the helix orientation of the coiled coil. [10][11][12][13][14][15] Besides the leucine zipper, other zippers such as an alanine zipper, 16 a tryptophan zipper, 17 and a phenylalanine zipper 18 also have been designed.…”

Crystal Structure of Inhibitor of Growth 4 (ING4) Dimerization Domain Reveals Functional Organization of ING Family of Chromatin-binding Proteins

“…Experimentally determined protein coiled-coil structures consist of two to six helices, with a single reported case of seven helices in a designed variant of the transcription factor GCN4 (34). The crystal structure of the ING4 N-terminal domain resembles the structure of the RNA-binding protein Rop (repressor of primer), the paradigm of four-helix antiparallel coiled coils (35), but with longer helices and loops and an additional two-turn-long ␣1 helix.…”