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New currency for old rope: from coiled-coil assemblies
to a-helical barrels
Derek N Woolfson1,2, Gail J Bartlett1, Marc Bruning1 and
Andrew R Thomson1
a-Helical coiled coils are ubiquitous protein–protein-interaction
domains. They share a relatively straightforward sequence
repeat, which directs the folding and assembly of amphipathic ahelices. The helices can combine in a number of oligomerisation
states and topologies to direct a wide variety of protein
assemblies. Although in nature parallel dimers, trimers and
tetramers dominate, the potential to form larger oligomers and
more-complex assemblies has long been recognised. In
particular, complexes above pentamer are interesting because
they are barrel-like, having central channels or pores with welldefined dimensions and chemistry. Recent empirical and rational
design experiments are beginning to chart this potential new
territory in coiled-coil space, leading to intriguing new structures,
and possibilities for functionalisation and applications.
Addresses
1
School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8
1TS, UK
2
School of Biochemistry, University of Bristol, Medical Sciences
Building, University Walk, Bristol BS8 1TD, UK
Corresponding author: Woolfson, Derek N (D.N.Woolfson@bristol.ac.uk)
Current Opinion in Structural Biology 2012, 22:1–10
This review comes from a themed issue on
Engineering and design
Edited by Jane Clarke and Bill Schief
0959-440X/$ – see front matter
# 2012 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.sbi.2012.03.002
Introduction: backdrop to and outlook for this
review
The concept of the a-helical coiled-coil protein structure—that is, two or more helices wrapped around each
other to form consolidated rope-like assemblies—has
been with us for nearly 60 years [1,2], experimental
validations of this for over a quarter of a century [3–5],
and the rational engineering and design of such structures
has been finessed considerably over the past two decades
[6,7]. By any measure, coiled-coil structural biology constitutes a mature research field. Nevertheless, coiled coils
continue to surprise and puzzle us. New coiled-coil
assemblies are coming to light that expand the repertoire
of structures observed [8]; imaginative and ambitious
design applications are being attempted with success
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[9]; and new analysis, modelling and prediction
methods are developing at pace [10–12,13,14,15]. Still,
there remains considerable scope for exploration in the
area: it is likely that other coiled-coil structures not yet
observed in nature, or realised through design are
possible; the outlook for de novo design, specifically with
respect to generating new protein functions is tantalising;
and, with improved understanding of sequence-to-structure relationships in coiled coils, the prospects for even
better prediction, design and engineering are promising.
This review attempts to capture some of this recent
activity in, and potential for the coiled-coil field.
Basics of coiled-coil sequence, structure and
function
a-Helical coiled coils constitute on average 3% of all
protein-encoding regions of the known genomes [16].
They were first recognised in certain classes of fibrous
protein, and, possibly as a result of this, their primary role
has mainly been considered as structural. Indeed, coiledcoil domains are largely associated with directing, specifying and cementing protein–protein interactions. However, they play oligomerisation and structural roles in
virtually all intracellular and extracellular processes, including transcription, ATP synthesis, intracellular transport, the cytoskeleton and cell structure, transmembrane
signalling, membrane fusion and remodelling, and the
formation and remodelling of the extracellular matrix.
Coiled coils also come in a wide variety of sizes, structural
types, and biochemical compositions.
As stated, a-helical coiled coils comprise two or more ahelices wrapped, or supercoiled, around each other to
form rope-like structures, Figure 1a. The helices are
amphipathic, being encoded by a relatively straightforward sequence repeat of hydrophobic (H) and polar (P)
residues, (HPPHPPP)n, known as the heptad repeat and
often designated abcdefg, Figure 1b; although there are
variations on this pattern, which essentially combine
different 3-residue and 4-residue spacings of hydrophobic
side chains. The helices combine to bury their hydrophobic a/d faces, Figure 1c. However, the mismatched
periodicities of the heptad repeat (with H-type residues
every 3.5 residues) and the a-helix (with 3.6 residues per
turn) results in this a/d seam winding slowly around the
helix; and, consequently, two or more helices wrap with a
left-handed supercoil to maximise burial of the H-type
side chains. Partly because the hydrophobic effect is not
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2 Engineering and design
Figure 1
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Current Opinion in Structural Biology
The basics of coiled-coil sequences and structures. (a) The rope-like structure formed by a relatively long coiled coil, the dimeric contractile protein
tropomyosin (PDB ID: 2EFR). (b) Helical-wheel diagram showing how the heptad repeat abcdefg tracks around a helical structure (in this case with 3.5
residues per turn). (c) One-heptad slice through the structure of the leucine-zipper region of the yeast transcriptional activator GCN4 (2ZTA). (d) A
Coiled-coil Basis Set comprising design dimer (CC-Di, left), trimer (CC-Tri, middle) and tetramer (CC-Tet, right, 3R4A). Oligomer-state specifying
asparagine side chains at heptad position a in CC-Di and d in CC-tri are shown in red and green, respectively. These three oligomer states represent
the major ones formed by the coiled-coil motif [10]. In panels a, c and d, colouring of the heptad positions, abcdefg, (and in C the side chains) follows
the CC+ standard for heptad positions (a = red; b = orange; c = yellow; d = green; e = cyan; f = blue; g = magenta) [10]. Structural images created
using PyMol (http://www.pymol.org).
specific, many different combinations of helices are
possible, including parallel, antiparallel, homo-typic
and hetero-typic arrangements, as well as various oligomerisation states [17–19], Figure 1d. This is the traditionally accepted, text-book model for coiled-coil sequenceto-structure relationships and assembly.
Knobs-into-holes packing and sharper
sequence-to-structure relationships
Of course, there must be more to sequence-to-structure
relationships in coiled coils to account for the large
proportion of such sequences in the known genomes,
Current Opinion in Structural Biology 2012, 22:1–10
the complexity of structures that they form, and the
presumed necessity for these to be orthogonal, that is,
non-promiscuous. The key to understanding this lies in
the knobs-into-holes (KIH) interactions first postulated
by Crick [1]. Side chains from adjacent helices in coiledcoil structures do not simply contact each other, rather
they interlock in specific and intimate ways: a side chain,
referred to as the knob, from one helix interdigitates within
a cluster of four side chains projecting in a diamondshaped hole from the partnering helix. For example in
parallel structures, an a knob coordinates within a dgad
hole, Figure 2a, b.
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a-Barrels Woolfson et al.
3
There are further subtleties. For example, the a and d
knobs of dimers project towards their partner helices in
different ways: the former projects out of the interface and
the latter directly into it, Figures 1b, c and 2b. Now
consider how the knob residues project from one helix
relative to another as oligomer state increases, Figure 2c:
they change; the angle that the knob makes with the base
of the hole is not only different for each knob site (a or d),
but also in each oligomer. These angles are referred to as
core-packing angles, but essentially for coiled-coil dimers,
trimers and tetramers they fall into three types: the
aforementioned packings at a and d of parallel dimers
are known as parallel and perpendicular, respectively; in
parallel tetramers these arrangements are swapped; and in
parallel trimers the packing at the two sites is somewhere
between these, and referred to as acute, Figure 2c [20].
Perhaps not surprisingly given the intimacy of KIH
interactions, these different shapes lead to different
amino-acid preferences [21].
influence of every amino acid in the sequence. We
generated three peptides with the key Harbury combinations of Ile and Leu at a and d as foregrounds: that is,
a = Ile, d = Leu (CC-pIL); a = d = Ile (CC-pII); a = Ile,
d = Leu (CC-pLI). The g–f backgrounds were also standardised to (EHAAHKX)4,a where: H is Ile or Leu as
stated, and X represents the outer f positions of the
heptad repeat, which were made combinations of Gln,
Lys or Tyr for solubility and to add a chromophore. CCpII and CC-pLI behaved as expected: they were trimeric
and tetrameric in solution, and their structures were
solved as parallel dimers and tetramers by X-ray crystallography, respectively, Figure 1d. Consequently, we
dubbed these CC-Tri and CC-Tet to signify that they
are characterised components of a Coiled-coil Basis Set.
Subsequently, CC-Tri has been used to nucleate the
assembly of an otherwise poor-folding bacterial collagen
[30], and CC-Tet has been mutated to explore larger
oligomer states (see below) [8].
This connection between KIH packing and sequence was
first noted by Harbury and colleagues in two classic
papers [20,22]. In these studies, variants of the leucinezipper peptide, GCN4-p1, taken from a yeast transcriptional activator, were made in which four consecutive
pairs of a and d sites were changed wholesale to combinations of Ile, Leu and Val. The key mutants were:
a = Ile, d = Leu (GCN4-pIL); a = d = Ile (GCN4-pII);
a = Ile, d = Leu (GCN4-pLI). These formed parallel
dimers, trimers and tetramers, respectively, and linked
core-packing angles to sequence: Leu is most tolerated at
perpendicular sites, and Ile (or Val) are preferred at
parallel or acute packings. Largely, these have been borne
out through both bioinformatics [19,21], and experimental studies [7].
Surprisingly, CC-pIL did not form a dimer as anticipated,
but a parallel trimer both in solution and the crystal state.
Others have also observed that the a = Ile, d = Leu foreground does not necessarily lead to parallel dimers [31].
These are important findings with implications for
protein engineering, design and prediction, but how
can they be reconciled with the earlier studies? Analysis
of parallel dimers and trimers from CC+ reveals that
although Leu is the most abundant of amino acids at
the a and d sites—indeed, this holds across all oligomer
states—it is not very discriminating in terms of oligomer
state. Ile and Val are selected against at the perpendicular-packing sites, and are favoured, though only slightly,
at the parallel-packing sites. Thus, the a = Ile, d = Leu
foreground does not strongly favour dimers. What does
appear to tip the balance further in natural sequences is a
small number of other specific amino-acid placements.
For example, Asn at a strongly favours parallel dimers,
which is a long-recognised relationship [32–34]. Indeed,
placing a single Asn at the third a site in CC-pIL causes a
complete and robust switch to parallel dimer to give the
Basis-set peptide CC-Di, Figure 1d.
Over the intervening two decades, many groups have
used either these GCN4-p1 variants directly, or the
principles for what to use at a and d in order to specify
oligomer state, in an impressive range of protein engineering and de novo design studies [7,23,24]. However,
nature and natural sequences are not so straightforward;
they have more variations, both in terms of amino-acid
usage and combinations. Moreover, a key question is, how
transferable are the Harbury relations to other, nonGCN4 or even entirely de novo systems?
On this point, recently we set out to create a toolkit of
reliable de novo designed coiled-coil peptides that might
be useful in protein engineering and synthetic biology
([25]; unpublished data). There were two primary reasons
for doing this de novo: First, although it has had considerable impact, the GCN4 system suffers from structural
plasticity; that is, small changes in sequence can lead to
large changes in structure as demonstrated by several
studies [26–29]. Second, in principle at least, the rationaldesign approach allows us to account for the structural
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New analysis, database, prediction and
modelling tools for coiled-coil structure
Thankfully, coiled-coil structures do not have to be
inspected by eye to uncover their secrets. Two programs
in particular garner many of the important parameters of
coiled-coil geometry. TWISTER [35] and SOCKET [19]
identify important structural features from 3D coordinates of coiled-coil structures, such as those deposited
in the RCSB Protein Data Bank (PDB) [36]. Whilst
TWISTER calculates backbone and related parameters
for coiled-coil geometries, SOCKET identifies runs of
a
Note the order here has been changed to gabcdef, which we find more
useful because of the potential for gh:eh+1 polar–polar interactions.
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4 Engineering and design
Figure 2
(b)
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a = parallel d = perpendicular
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Type N (xHxxHxx)
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51º
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Current Opinion in Structural Biology
Knobs-into-holes (KIH) interactions. (a) Crick’s original helical-net concept, which led to the idea of KIH packing. On the left-hand side, the heptad
repeats for two helices (coloured red and blue) are projected onto flat surfaces. These illustrate how the hydrophobic a/d seams wind around the
helices in the opposite sense to that of a regular, right-handed a-helix. On the right-hand side, the two nets are interlaced to show how each residue on
Current Opinion in Structural Biology 2012, 22:1–10
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a-Barrels Woolfson et al.
KIH interactions between a-helices, noting patterns
within these. In this way, SOCKET assigns heptad patterns, oligomer states and topology, and it also provides
quantitative measures of core-packing angles. Recently,
SOCKET has been applied to the whole of the PDB
to create a relational database of coiled-coil structures,
CC+ [10], http://coiledcoils.chm.bris.ac.uk/ccplus/search/
. CC+ may be searched in a variety of ways—based on
keywords, structural properties, sequence and interactions—to return subsets of coiled-coil structures, and
various data from these can be exported in a number of
formats. Within these outputs, amino-acid profiles—that
is, tables giving the proportions of each amino acid at the
abcdefg sites—are particularly useful as they address the
aforementioned issue of more-diverse amino-acid usage,
and provide a basis for developing new prediction algorithms and de novo designs. Also via the same URL, the
Periodic Table of Coiled-coil Architectures organises the
known structures according to oligomer state (the columns or groups of the table) and complexity of the
arrangements (the rows or periods) [18]. Users should
be aware, however, that whilst CC+ is regularly updated
the Periodic Table of Coiled Coils is not.
Resources such as SOCKET and CC+ provide unprecedented links between coiled-coil sequence and structure,
and thus open possibilities for protein-structure prediction, engineering and design. For example, the SCORER
algorithm [21], which predicts propensities of sequences
to form coiled-coil dimers and trimers has recently been
retrained using structurally verified coiled coils in CC+
[12]; similarly PairCoil [37] and MultiCoil [38], sequencebased coiled-coil assignment and oligomeric-state predictors respectively, have been retrained on sets including sequences derived from SOCKET analyses of
the PDB [15,39]. In addition, PrOCoil [14] uses
SOCKET-derived information to train a support-vector-machine classifier of dimeric and trimeric coiled-coil
sequences. A significant improvement on coiled-coil coverage in prediction has been achieved with a new fourstate predictor for distinguishing antiparallel and parallel
dimers, and parallel trimers and tetramers, LOGICOIL
(unpublished method), which covers >90% of known
coiled-coil structures. Using data from CC+ and hidden
Markov models implemented in SUPERFAMILY [40],
5
an annotation of coiled coils in all completely sequenced
genomes has led to a genome-wide evaluation of coiledcoil evolution and homology based prediction called
SPIRICOIL [16].
Related to the prediction problem, Gellman and colleagues have compared experimental data for a model
antiparallel coiled-coil system and correlations culled
from CC+. Specifically for this class of structure, they
find that high-order interactions in a0 -a-a0 vertical triads
contain information that favours certain helix-helix pairings [41], notably hetero-combinations such as Ile–Leu–
Ile are favoured; whereas, residue combinations at d0 -d-d0
are less discriminating [42]. These studies are important,
demonstrating that coiled-coil prediction, modelling and
design needs to move past amino-acid profiles and pairwise correlations, and tackle the problem more holistically including larger constellations of side chains.
Keating’s group and others are also developing methods
that bridge the computational and experimental aspects
of coiled-coil prediction and design, particularly in the
areas of antiparallel coiled-coil formation [43], and the
definition of hetero-specific parallel coiled-coil dimers
[44–46]; both of which are important areas that are far
from fully understood.
New modelling methods, which build on foregoing work
[1,47–49], are being developed for coiled-coil structures
with both prediction and design in mind. For example,
Grigoryan and DeGrado present a method of parameterizing coiled coils using modified Crick equations that allow
for parallel, antiparallel and mixed orientations as well as
helical sliding along the interfacial axis [13]. Using this
approach, the vast majority of natural coiled-coil structures
were found to be within 1 Å RMSD of idealised ‘Crick’
backbones. Parameters derived from known coiled coils
demonstrate that the geometrical space occupied by coiled
coils is severely restricted: parallel and antiparallel structures have distinct and limited axial offset; likewise superhelical frequency and pitch angle are limited among all
natural coiled coils regardless of orientation and oligomeric
state; there is a linear relationship between the superhelical
radius and oligomerisation state, with residues at the a and
d positions showing distinct propensities at different superhelical radii in parallel coiled coils. This work is important
( Figure 2 Legend Continued ) one helix effectively interdigitates between four residues of the partnering helix. This leads naturally to the left-handed
supercoil of the coiled coil. (b) Examples from the X-ray crystal structure of GCN4-p1, a parallel, homodimeric coiled coil (PDB ID: 2ZTA): top, an a
knob (red) coordinating within a dgad hole (blue); bottom, a d knob and an adea hole. (c) Typical core-packing angles made by a and d knobs in
parallel dimers (uppermost, 2ZTA), trimers (middle, 1AQ5) and tetramers (lowermost, 2GUS). (d) From top to bottom: the standard N-type heptad
repeat, and its structural realisation at a parallel dimer interface (2ZTA); type I repeat partially observed in trimers (1AQ5); type II repeat, tetramers–
hexamers (3R3K); and the type III repeat anticipated above heptamer (1EK9). The angles shaded green in the bottom three helical-wheel diagrams are
idealised and represent those subtended between the two hydrophobic seams of the types I–III repeats. In both type II and type III the interface is
extendable: the residues involved in forming this interface are picked out in dots, and the region of the interface is shown on the structure and helicalwheel diagrams with a solid black line. Note that the structural example of the type III repeat comes from an antiparallel coiled-coil assembly: the
central helix follows an N-to-C-terminal path into the page, whilst the two outer helices follow a C-to-N terminal path into the page; and the
juxtaposition of heptad positions making knobs-into-holes interactions is altered. Colouring follows the CC+ standard for heptad positions (a = red;
b = orange; c = yellow; d = green; e = cyan; f = blue; g = magenta) [10]. Structural images created using PyMol (http://www.pymol.org).
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6 Engineering and design
Figure 3
(a)
(d)
(b)
(e)
(c)
(f)
(g)
Current Opinion in Structural Biology
Orthogonal views of a-helical barrels. (a) The CC-Hex hexamer with a 5–6 Å channel (PDB ID: 3R3K). (b) The L24D:L24H heterohexamer (3R48). (c)
The heptameric mutant of GCN4-p1, which has an 9 Å channel (2HY6). (d) The TolC structure, which has a periplasmic 12-helix a-barrel with a 25 Å
pore (1EK9). (e) The Wza assembly with an 8-helix transmembrane barrel and a 17 Å internal diameter (2J58). (f) The cytotoxin ClyA with a >50 Å pore
contributed to by 24 helices (2WCD). (g) The open state of the heptameric mechanosensitive channel MscS with a 13 Å channel (2VV5). The a-helical
barrel structures are shown in colour.
in narrowing down parameter and, therefore, structural
space available to coiled coils, and, so, paves the way
to better model building for prediction and design applications. A web site, CCCP (http://arteni.cs.dartmouth.edu/
cccp/), is available for generating idealised Crick backbones using these methods.
Current Opinion in Structural Biology 2012, 22:1–10
Extended KIH packing and the potential for
higher-order states
Another consequence of increasing oligomer state is that
residues other than those at a and d become more involved
in the helix-helix interfaces, Figure 2c, d. Specifically, the e
and g sites become progressively buried. The extreme is
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a-Barrels Woolfson et al.
that residues at these positions become knobs, and part of
what have been termed peripheral KIH interactions [50].
These trends can be seen through SOCKET analyses of
the GCN4 variants, the Basis-set peptides and natural
coiled coils. The natural extremes are provided by a small
number of pentamers in the CC+ database, which include
COMP, phospholamban, a rotavirus enterotoxin, CorA,
and some engineered structures with aromatic cores [51–
56]. In these structures many of the g, a, d and e positions act
as knobs, and some of the sequences approximate to
HHxxHHx repeats, rather than more traditional xHxxHxx
heptad repeats. (Note the gabcdef order.) This relates back
to an idea of offset double heptad repeats [50], which had been
considered previously, at least in theory, by several groups
[57–59].
The above HHxxHHx pattern can be split up to give two
heptad repeats, xHxxxHxx plus HxxxHxx, both of which
are 3,4-repeats of H-type residues. This is one of three
possible offset double-heptad repeats, the others being
HHxxHxx and xHxHHxH, Figure 2d [50]. For simplicity,
we refer to these and the original heptad pattern as:
xHxxHxx, N; HHxxHxx, I; HHxxHHx, II, and xHxHHxH,
III. These are arranged in the order of increasing oligomer-state that they give rise to: dimers best fit N; I is
observed in trimers; II in tetramers and pentamers; and
III opens up possibilities for a range of complex coiled
coils, including a-helical sheets and a-helical barrels,
Figure 2d [50,57–60]. These are not hard and fast rules,
but we find the nomenclature and concepts useful.
What’s more, they question what is meant and understood by ‘coiled coil’ both in terms of sequence and
structure, and what other structures might be accessible
to this often-considered straightforward domain.
New higher-order assemblies, a coiled-coil
hexamer (CC-Hex)
Considering these possibilities of offset double-heptad
repeats opens up a whole new space in coiled-coil assembly that is just beginning to be realised and explored. For
example, recently, the Basis-set tetramer, CC-Tet, has
been used to create a parallel coiled-coil hexamer, CCHex [8]. The details of this study are illuminating. First,
CC-Tet has a traditional N-type heptad repeat that specifies tetramer using the Harbury relationships, that is
(ELAAIKX)4. The X-ray structure reveals a parallel tetramer with classical perpendicular and parallel KIH interactions at a and d, respectively. However, and consistent
with a type-II pattern: 1/4 of the total number of KIH
interactions identified by SOCKET fall at the g and e
sites. The sequence was mutated to exchange all of Lys at
e with the Ala at b, to give the following repeat, (ELKAIAX)4, which potentially broadens the hydrophobic
seam to a, d and e. The new peptide forms a very stable
a-helical hexameric complex in solution, which crystallises as a parallel hexamer, Figure 3a, [8]. This structure
had not been observed before; indeed, on the basis of
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7
likely overstretched KIH interactions, it had been predicted that it should not exist [50]. Nonetheless, the new
structure, CC-Hex, tests positive as a 6-helix parallel
coiled coil in both TWISTER and SOCKET, and all
of the residues at g, a, d and e act as knobs in classical KIH
interactions. However, and somewhat consistent with the
earlier prediction, the core-packing angles at the g and a
sites fall on the periphery of the distribution observed for
all perpendicular sites of parallel dimers tetramers in
CC+; n.b., those at d and e fall in the middle of the range
observed for parallel packing in the same structures.
CC-Hex is intriguing for another reason, which is likely to
have a much broader impact [61]: though the six helices
come together by largely hydrophobic contacts they leave
a central well-defined channel of 5–6 Å, Figure 3a. This is
lined exclusively by methyl groups of the Leu and Ile side
chains at a and d. Nonetheless, there is electron density
within the channel, which is adequately modelled as a
disrupted chain of water molecules. The side chains of
Leu-24—the a site of the third heptad, and which point
directly into the channel—are mutable, accepting residues as diverse as aspartic acid and histidine. These
L24D and L24H mutants, although destabilised, form
parallel hexamers in solution and the crystal state. Moreover, they combine to form an intriguing heterohexamer
with alternating Asp-containing and His-containing
chains, Figure 3b.
Towards a-helical barrels
CC-Hex raises the bar of oligomer states accessible to
coiled-coil motifs. So how far can the concept of
extended KIH patterns be pushed to model, predict
and design new coiled-coil oligomers? CC-Hex is a true
and classical coiled coil; that is, it has a contiguous ring
of KIH interactions, albeit surrounding an internal
channel. There is heptameric coiled coil in the CC+
database—a mutant of GCN4-p1 with g = e = Ala [28]—
however we note that this is far from a classical coiled
coil: the SOCKET assignments of KIH interactions and
heptad register are not straightforward; and the helices
spiral rather than packing blunt-ended, with the first
and seventh helices offset by a whole heptad,
Figure 3c. Moreover, an ideal sequence pattern for a
heptameric coiled coil falls midway between the typeII and III patterns. In these respects, the heptamer
might be regarded as the tipping point between classical
and more-complex coiled coils [8]. Nevertheless, the
structure is barrel-like, with the internal channel is fully
occupied by hexane-1,6-diol co-solvent, and the structure is interesting further and potentially useful for
that.
The next oligomer state represented in the PDB and CC+
is a dodecamer that is part of the multidrug efflux protein
TolC from E. coli [60], Figure 3d. This is a beautiful,
multifunctional, but complicated structure. The central,
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8 Engineering and design
periplasm-spanning domain comprises 12 antiparallel ahelices arranged in a barrel; indeed, to our knowledge,
this was where the term a-barrel was first coined [60]. The
antiparallel topology leads to some unwinding of the
coiled coil [58]. Nevertheless, the helices do show some
KIH interactions in SOCKET, and these fit the type-III
pattern, Figure 2; effectively the structure is a ring of
helices connected via two dimer-like hydrophobic seams
per helix.
Interestingly, recent X-ray and cryoEM studies of the
viral genome-delivery portal from bacteriophage P22
reveal a long 12-helix barrel, proposed to be responsible
genome ejection into host cells [62,63]. At this stage the
resolution of this part of the structure is insufficient to test
if the helices are cemented by KIH interactions. Nonetheless, it would appear the 12-helix barrel of TolC is not
unique, that is, a singleton in structural terms.
In addition, to rings of 12 helices, there is a growing class of
membrane-spanning and membrane-associated a-helical
barrels that suggest that various pore and channel sizes can
be achieved, Figure 3e–g. For example, the first membrane-spanning a-helical barrel was reported in the structure of Wza [64]. Wza is an outer-membrane lipoprotein
from E. coli. It forms part of the polysaccharide export
machinery essential to capsule formation around the bacterium. Domain 4 of the protein is an amphipathic a-helix
that octomerises in the membrane to produce a polar
channel with an internal diameter of 17 Å. A much larger
barrel has been observed in the a-pore-forming toxin ClyA
from E. coli and Salmonella [65]. The active form results
from a large structural rearrangement of the protein in
which 12 three-helix bundles combine to produce the
barrel. Each bundle contributes two helices to the innerface of barrel, leading to a negatively charged, cationselective pore with an internal diameter of 70 Å. Finally,
the transmembrane region of the mechanosensitive channel, MscS, again from E. coli, comprises a heptamer of
three-helix bundles [66,67]. In this case the first two helices
buttress the third, which forms a much tighter and controllable channel. It is proposed [67] that the conformations
of the first and second helices respond to changes in
osmotic pressure on cells by twisting and thus opening
up the channel from 5 Å to 13 Å in its closed [66] and
open states [67], respectively. In terms of coiled-coil
structures, however, only part of the large ClyA barrel tests
positive in SOCKET, Figure 3f.
Conclusions: prospects for the future
discoveries and designs
The leap from 6-membered and 7-membered barrels to
those with 12 helices raises the questions: are true coiledcoil octomers, nonomers, decamers and hendecamers
possible, and what about oligomers above that? If any
of these were to be observed in nature, or could be
designed de novo, they would not only be of interest in
Current Opinion in Structural Biology 2012, 22:1–10
coiled-coil ‘stamp collecting’, but could have an impact in
the design of ion-channels, binding proteins, sensors and
even enzymes [8,61]. To illustrate this potential, an
elegant computational redesign—using the aforementioned methods from Grigoryan and DeGrado [13]—of
a previous de novo coiled-coil bundle has successfully
produced a peptide that binds and solubilises carbon
nanotubes [9]; interestingly, the designed bundle is
for an antiparallel 6-helix coiled coil, though confirmation
of this awaits high-resolution structural studies.
Accessing the various other ‘largermers’, however, presents
a number of challenges, including: the apparent requirement for very hydrophobic peptides to satisfy the
xHxHHxH, type-III pattern; residue selection at the H
sites; and the aforementioned problem with unwinding the
coiled coils. Even with these tackled it may be that
specifying particular oligomer states will be difficult, as
the difference in energy between alternate states is
expected to become smaller as oligomer size increases.
Indeed, on purely geometric grounds, the type-III pattern
should lead to a 14-mer, which has yet to be observed
[57,59], rather than the dodecamer that is realised in TolC
[60]; though of course, other forces are almost certainly at
play in the folding of this complex protein structure.
Further on this point, the TolC structure has been analysed
theoretically to understand its physics and sequence-tostructure relationships [58], but, to our knowledge, these
have not led to a successful de novo design for a dodecamer.
Thus, it is almost certain that further developments in our
current theory, sequence-to-structure relationships and
methods for modelling coiled-coil proteins will be required
to designed versions of the dodecamer, and, indeed any
other largermer states above hexamer. Nonetheless, with
the new activities in all three of these areas, and the new
structures that have been realised experimentally, the
outlook for coiled-coil research leading to better understanding and functional designs is promising.
Acknowledgements
We thank the Woolfson group, Leo Brady, Vince Conticello, Stuart Conway,
Noah Linden and Richard Sessions for helpful discussions, and the BBSRC
and EPSRC of the UK for funding (grant numbers: BB/G008833/1 and EP/
J01430/1).
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