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This year marks the 50th anniversary of Crick's seminal paper on the packing of alpha-helices into coiled-coil structures. The central tenet of Crick's work is the interdigitation of side chains, which directs the helix-helix interactions; so called knobs-into-holes packing. Subsequent determinations of coiled-coil-protein sequences and structures confirmed the key features of Crick's model and established it as a fundamental concept in structural biology. Recently, we developed a program, SOCKET, to recognise knobs-into-holes packing in protein structures, which we applied to the Protein Data Bank to compile a database of coiled-coil structures. In addition to classic structures, the database reveals 4-helix bundles and larger helical assemblies. Here, we describe how the more-complex structures can be understood by extending Crick's principles for classic coiled coils. In the simplest case, each helix of a 2-stranded structure contributes a single seam of (core) knobs-into-holes to the helical interface. 3-, 4-, and 5-Stranded structures, however, are best considered as rings of helices with cycles of knobs-into-holes. These higher-order oligomers make additional (peripheral) knobs-into-holes that broaden the helical contacts. Combinations of core and peripheral knobs may be assigned to different sequence repeats offset within the same helix. Such multiple repeats lead to multi-faceted helices, which explain structures above dimers. For instance, coiled-coil oligomer state correlates with the offset of the different repeats along a sequence. In addition, certain multi-helix assemblies can be considered as conjoined coiled coils in which multi-faceted helices participate in more than one coiled-coil motif.

Coiled-coil motifs foster a wide variety of protein– protein interactions. Canonical coiled coils are based on 7-residue repeats, which guide the folding and assembly of amphipathic -helices. In many cases such repeats remain unbroken for tens to hundreds of residues. However, the sequences of an increasing number of putative and characterised coiled coils digress from this pattern. We probed the consequences of nonheptad inserts using a designed leucine-zipper system. The parent peptide, SKIP0, which had four contiguous heptads, was confirmed as a parallel homodimer by circular dichroism spectroscopy and analytical ultracentrifugation. Seven daughter peptides were constructed in which 1 to 7 alanine residues were inserted between the central heptads of SKIP0. Like SKIP0, SKIP7 formed a stable helical dimer, but the other peptides were highly destabilised, with the order of dimer stability SKIP4 SKIP5 > SKIP6 > SKIP3 > SKIP2 > SKIP1. These results are consistent with an extended theory of coiled-coil assembly in which coiled-coil-compatible motifs are based on 3- and 4-residue spacings and most notably heptad (7-residue) and hendecad (11-residue) repeats. Thus, they help explain why in natural sequences, inserts after canonical heptad repeats most commonly comprise 4 residues. Possible biological roles for nonheptad inserts are discussed. © 2002 Elsevier Science (USA)

The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions. Coiled coils comprise two or more alpha-helices that wind around each other to form "supercoils". The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat. Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states. To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures. We used SOCKET to gather a set of unambiguous coiled-coil structures from the RCSB Protein Data Bank. Rather than searching for sequence features, the algorithm recognises the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains. SOCKET unambiguously defines coiled-coil helix boundaries, oligomerisation states and helix orientations, and also assigns heptad registers. Structures retrieved from the Protein Data Bank included parallel and anti-parallel variants of two, three and four-stranded coiled coils, one example of a parallel pentamer and a small number of structures that extend the classical description of a coiled coil. We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction and design. To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino acid profiles for the heptad repeats of different motifs.

The coiled coil is a ubiquitous protein-folding motif. It generally is accepted that coiled coils are characterized by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic alpha-helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed "knobs-into-holes packing". We developed software, SOCKET, to recognize this packing in protein structures. As expected, in a trawl of the protein data bank, we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, we identified structures with multiple, overlapping heptad repeats. This observation extends Crick's original postulate: Multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to the coiled-coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by approximately 150 degrees -160 degrees around the alpha-helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multihelix assemblies. Accordingly, we refer to these two forms as alpha-sheets and alpha-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.

Cytoplasmic dynein is the major minus end-directed microtubule motor in eukaryotes. However, there is little structural insight into how different cargos are recognized and linked to the motor complex. Here we describe the 2.2 Å resolution crystal structure of a cargo-binding region of the dynein adaptor Bicaudal-D (BicD), which reveals a parallel coiled-coil homodimer. We identify a shared binding site for two cargo-associated proteins—Rab6 and the RNA-binding protein Egalitarian (Egl)—within a region of the BicD structure with classical, homotypic core packing. Structure-based mutagenesis in Drosophila provides evidence that occupancy of this site drives association of BicD with dynein, thereby coupling motor recruitment to cargo availability. The structure also contains a region in which, remarkably, the same residues in the polypeptide sequence have different heptad registry in each chain. In vitro and in vivo analysis of a classical Drosophila dominant mutation reveals that this heterotypic region regulates the recruitment of dynein to BicD. Our results support a model in which the heterotypic segment is part of a molecular switch that promotes release of BicD autoinhibition following cargo binding to the neighboring, homotypic coiled-coil region. Overall, our data reveal a pivotal role of a highly asymmetric coiled-coil domain in coordinating the assembly of cargo–motor complexes.

In recent years, a number of bacterial coiled-coil proteins have been characterised which have roles in cell growth and morphology. Several have been shown to have a cytoskeletal function and some have been proposed to have an IF-like character in particular. We recently demonstrated in Streptomyces coelicolor a cytoskeletal role of Scy, a large protein implicated in filamentous growth, whose sequence is dominated by an unusual coiled-coil repeat. We present a detailed analysis of this 51-residue repeat and conclude that it is likely to form a parallel dimeric non-canonical coiled coil based on hendecads but with regions of local underwinding reflecting highly periodic modifications in the sequence. We also demonstrate that traditional sequence similarity searching is insufficient to identify all but the close orthologues of such repeat-dominated proteins, but that by an analysis of repeat periodicity and composition, remote homologues can be found. One clear candidate, despite a great size discrepancy and unremarkable sequence identity, is the known filament-former FilP in the same species. Both proteins appear distinct from the archetypal bacterial IF-like protein; they therefore may constitute a new class of bacterial filamentous protein. The similar sequence characteristics of both suggest their likely oligomer state and a possible mechanism for higher-order assembly into filaments. Another remote homologue in Actinomyces was highlighted by this method. Further, a known coiled-coil protein, DivIVA, appears to share some of these sequence characteristics.

The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a seven-residue (heptad) sequence repeat. The positions of this repeat are labelled a-b-c-d-e-f-g, with residues at a and d tending to be hydrophobic. Such sequences form amphipathic α-helices, which assemble into helical bundles via knobs-into-holes interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank. Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help to explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues, i.e. a/f′-b/g′-c/a′-d/b′-e/c′-f/d′-g/e′. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open 'α-sheets' and closed 'α-cylinders'. We highlight these with descriptions of natural structures and outline possibilities for protein design.