Computational Prediction and Design for Creating Iteratively Larger Heterospecific Coiled Coil Sets

Abstract

A major biochemical goal is the ability to mimic nature in engineering highly specific protein-protein interactions (PPIs). We previously devised a computational interactome screen to identify eight peptides that form four heterospecific dimers despite 32 potential off-targets. To expand the speed and utility of our approach and the PPI toolkit, we have developed new software to derive much larger heterospecific sets (≥24 peptides) while directing against antiparallel off-targets. It works by predicting T values for every dimer on the basis of core, electrostatic, and helical propensity components. These guide interaction specificity, allowing heterospecific coiled coil (CC) sets to be incrementally assembled. Prediction accuracy is experimentally validated using circular dichroism and size exclusion chromatography. Thermal denaturation data from a 22-CC training set were used to improve software prediction accuracy and verified using a 136-CC test set consisting of eight predicted heterospecific dimers and 128 off-targets. The resulting software, qCIPA, individually now weighs core a-a' (II/NN/NI) and electrostatic g-e' (EE/EK/KK) components. The expanded data set has resulted in emerging sequence context rules for otherwise energetically equivalent CCs; for example, introducing intrahelical electrostatic charge blocks generated increased stability for designed CCs while concomitantly decreasing the stability of off-target CCs. Coupled with increased prediction accuracy and speed, the approach can be applied to a wide range of downstream chemical and synthetic biology applications, in addition more generally to impose specificity on structurally unrelated PPIs.

Structural Determinants of Coiled Coil Mechanics

“…As a result, they are increasingly used as templates for protein design and sequences with a pre-determined thermodynamic stability can now be synthesized de novo. [3][4][5][6] Such sequences find application in peptide-based hydrogels [7][8][9][10] and protein origami structures 11,12 as well as in biosensors 13 and drug delivery systems. 8,[14][15] Considering their natural role as a mechanical scaffold, surprisingly little information is available about the sequence-structure-mechanics relationship of CCs.…”

Structural determinants of coiled coil mechanics

“…As a result, they are increasingly used as templates for protein design and sequences with a pre-determined thermodynamic stability can now be synthesized de novo. [5][6][7][8] Such sequences find application in peptide-based hydrogels [9][10][11] and protein origami structures 12,13 as well as in biosensors 14 and drug delivery systems. 15 Considering their natural role as a mechanical scaffold, surprisingly little information is available about the sequence-structuremechanics relationship of CCs.…”

Coiled coil protein origami: from modular design principles towards biotechnological applications

“…Recently, Crooks et al developed the largest CC set so far. 52 Using the bCIPA algorithm 53 a set of 8 parallel heterodimers was constructed comprising 4 heptad repeats. However, T m measurements revealed that only 7 pairs behaved as designed with T m 4 70 1C and at least a 10 1C gap before the most stable off-target interaction.…”

“…Firstly, the explored sequence space is restrained in accordance with previous rules discovered to govern CC oligomerization and pairing specificity. In the previously mentioned examples concerning the design of parallel CC sets, 47,48,52 only lysine and glutamic acid were allowed at e:g positions, while the a heptad positions could be occupied either by asparagine or isoleucine and at d positions only leucine was allowed. Only a, d, e and g heptad positions were subjected to design, while b, c, and f positions were occupied by helicity promoting amino acids.…”