Deriving Heterospecific Self-Assembling Protein–Protein Interactions Using a Computational Interactome Screen

Abstract

Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein–protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology.

Modular assembly of a protein nanotriangle using orthogonally interacting coiled coils

“…23 SYNZIPs have been described 19 , 20 , and binding pairs such as 10 : 22 and 17 : 18 could be useful as additional modules because their interactions are reported as stronger than potentially competing associations with any of SYNZIPs 1–6. Furthermore, synthetic coiled coils beyond SYNZIPs, as well as large sets of coiled - coil homo- and heterodimers from animal bZIP transcription factors that have been comprehensively tested for associations provide candidates for extended sets of orthogonal coiled coils 17 , 18 , 23 , 37 , 38 . With larger sets of coiled-coil modules that display strong interaction preferences, our approach may be extended to design more complex structures that can potentially be linked together in 2- or 3-dimensions, functionalized, attached to surfaces or incorporated with other designed elements into increasingly complex nanoassemblies.…”

“…Based upon a rich understanding of coiled-coil sequence-structure relationships that has been obtained over the past ~25 years, de novo designed coiled-coil toolkits, i.e. sets of coiled-coil units with known interaction properties, have been generated by using systematic search and/or rational 17 , 18 or computational approaches 19 – 23 . Because of their utility as short self-assembling protein modules, coiled coils have been used to construct a variety of assemblies, including fibers 24 , cages 25 , 26 , and nanotubes 27 with extended and geometrically irregular structures 28 .…”

Programmable design of orthogonal protein heterodimers

“…Creation of new specificities starting from naturally occuring interacting proteins, such as toxin/antidote pairs 7 , by sequence redesign has been difficult, often resulting in promiscuous binding 8 ; the natural specificity results at least in part from complementary variation in backbone conformation 9 . Orthogonal sets of 2–4 interacting coiled coil pairs have been created and experimentally validated 10,11 , including the widely used SYNZIPs 12–18 , but interaction promiscuity has again hampered the design of larger orthogonal sets.…”

Combined computational and intracellular peptide library screening: towards a potent and selective Fra1 inhibitor

“…These provide greater scope for stabilization of antagonist-target heterodimeric complexes, destabilize antagonist homodimers, and enhance overall interaction specificity. 25,[30][31][32] Residues within the Fra1 core are atypical and less favourable for hydrophobic interaction; however, Fra1W displays a hydrophobic core, consisting of I, V, as well as smaller A sidechains, to establish favourable interactions with Fra1. Reassuringly, in combination with the electrostatic component, the permutation of these aliphatic hydrophobics within Fra1W does not disproportionately favour interaction as a homodimer or with cJun.…”

“…The library was next screened using isCAN software based on the bCIPA algorithm, which has been described in detail elsewhere. [20][21][22]25,34 Briefly, the software provides a qualitative rank of affinity by estimating the thermal melting point (T m ) of every potential dimeric interaction within the system. In this library, every g and e position within the coiled coil, which are critical in forming electrostatic contacts within a coiled-coil sequence 19 were semirandomized to generate Q/E/K options, with a view to generating both potential attractive and repulsive options with the corresponding positions of the target (Fig.…”