Deriving Heterospecific Self-Assembling Protein–Protein Interactions Using a Computational Interactome Screen. Issue 2 (29th January 2016)

• Record Type: Journal Article
• Authors: Crooks, Richard O.; Baxter, Daniel; Panek, Anna S.; Lubben, Anneke T.; Mason, Jody M.
• Is Part Of: Journal of molecular biology. Volume 428:Issue 2 Part A(2016:Jan. 15)
• Page Start: 385
• Page End: 398
• Publication Date: 2016-01-29
• Subjects: PPI protein–protein interaction -- bCIPA bZIP coiled-coil interaction prediction algorithm -- SEC size-exclusion chromatography -- MS mass spectrometry; protein–protein interactions -- de novo design -- heterospecific proteins -- peptide -- interactome screen; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.11.022
• Languages: English
• ISSNs: 0022-2836

Abstract

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 T m values and (iii) used predicted T m 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. Graphical abstract: Highlights: Naturally occurring protein–protein interactions (PPIs) are highly specific. For designed PPIs, however, specificity can be notoriously difficult to engineer. We have computationally screened a vast interactome to derive four heterospecific PPIs. Eight peptides form four heterospecific coiled coils; all 32 off targets are disfavoured. The method can derive larger and increasingly complex sets of heterospecific PPIs