New currency for old rope: from coiled-coil assemblies to α-helical barrels

Woolfson, Derek N.; Bartlett, Gail J.; Bruning, Marc; Thomson, Andrew

doi:10.1016/j.sbi.2012.03.002

Cited by 133 publications

(158 citation statements)

References 68 publications

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“…These rules have been validated by database studies, 10,14 and are used as the cornerstone of much coiled-coil design. 2,[15][16][17] In addition, charged residues at e and g, which flank the hydrophobic core, Figure 1C&D, often form salt bridges in natural systems. 2,16,18,19 This is also exploited in coiled-coil design: judicious placement of complementary charges-e.g., Lys-Glu pairs at e and g-can be used to pattern sequences to control homotypic or heterotypic assembly of peptides otherwise possessing the same core a & d residues.…”

Section: Introductionmentioning

confidence: 99%

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

Fletcher¹,

Bartlett²,

Boyle³

et al. 2017

ACS Chem. Biol.

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The α-helical coiled coil is one of the best-studied protein–protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix–helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.

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Section: Introductionmentioning

confidence: 99%

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

Fletcher¹,

Bartlett²,

Boyle³

et al. 2017

ACS Chem. Biol.

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“…As a result, they are best described as -helical barrels. 32,33 Recently, we have reported both the serendipitous discovery, 34 and the rational, computational design of a series of -helical barrels, including pentamers, hexamers and a heptamer. 35 These structures have contiguous, mutable and accessible central channels, 36 which provide a basis for the installation of functional residues with precision and within defined environments.…”

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confidence: 99%

Installing hydrolytic activity into a completely de novo protein framework

et al. 2016

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Designing enzyme-like catalysts tests our understanding of sequence-tostructure/function relationships in proteins. Here, we install hydrolytic activity predictably into a completely de novo and thermo-stable -helical barrel, which comprises 7 helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses pnitrophenyl acetate with catalytic efficiencies matching the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.(134 words)

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“…Coiled-coil structures are widespread in the biological world [5], and keratins represent simple examples to illustrate the forces that bind these structures. Sequence based prediction of keratin structural domains can be taught as an illustrative example of the usefulness of protein structure prediction methods.…”

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confidence: 99%