Combining Constrained Heptapeptide Cassettes with Computational Design To Create Coiled-Coil Targeting Helical...

A total of 32 heptapeptides have been synthesized and characterized to establish the effect of K → D (i → i + 4) lactamization upon their ability to adopt a helical conformation. Because most parallel and dimeric coiled-coil sequences can be deconvoluted into gabcdef repeats, we have introduced fixed solvent exposed b → f (K → D) constraints into this design scaffold. Interfacial " a" hydrophobic (L/I/V/N) and " e/g" electrostatic (E/K) options (4 × 2 × 2 = 16 cassettes) were introduced as core drivers of coiled-coil stability and specificity. All present as random coils when linear but adopt a helical conformation upon lactamization. Helicity varied in magnitude from 34 to 68%, indicating different levels of constraint tolerance within the context of a sequence required to be helical for function. Using the oncogenic transcription factor cJun as an exemplar, we next utilized our bCIPA coiled-coil screening engine to select four cassettes of highest predicted affinity when paired with four gabcdef cassettes within the full-length cJun target counterpart (16⁴ = 65 536 combinations). This information was coupled with observed helicity for each constrained cassette to select for the best balance of predicted affinity when linear and experimentally validated helicity when constrained. As a control, the same approach was taken using cassettes of high predicted target affinity but with lower experimentally validated helicity. The approach provides a novel platform of modular heptapeptide cassettes experimentally validated and separated by helical content. Appropriate cassettes can be selected and conjugated to produce longer peptides, in which constraints impart appropriate helicity such that a wide range of targets can be engaged with high affinity and selectivity.