Biotechnology Letters

, Volume 41, Issue 1, pp 79–90 | Cite as

Clustered complementary amino acid pairing (CCAAP) for protein–protein interaction

  • Christina Kyung Eun Baek
  • Chang-Ho BaekEmail author
Original Research Paper



Designing a polypeptide sequence to interact with a preselected target polypeptide sequence of a protein has long been of interest, yet remains an elusive goal.


Here, we propose a novel concept named “Clustered Complementary Amino Acid Pairing (CCAAP),” which plays an essential role in protein–protein interaction (PPI). Complementary amino acid pairing (CAAP) is a pairing between two amino acids encoded by a codon and its reverse complementary codon. CAAP interactions largely agree with the physicochemical and stereochemical requirements for probable amino acid pairings. Interestingly, 82 PPI structure data revealed that clusters of CAAP interactions (CCAAP boxes) are predominantly found in all PPI sites. Analysis of all amino acid pairings in the CCAAP boxes unveiled amino acid-pairing preferences and patterns for PPI that allowed us to develop a new method for designing an oligopeptide sequence to bind to a chosen polypeptide sequence of any target protein.


Discoveries in the present study provide proof of the CCAAP principle.


Clustered complementary amino acid pairing Protein detection Protein–protein interaction Protein targeting Recombinant antibody Synthetic antibody Synthetic biology 



The authors acknowledge that the full financial support for this research received directly from Peption LLC (San Diego, CA, USA).

Supporting information

Supplementary Fig—1 Expression vectors for the production of the recombinant antibodies (rAbs).

Supplementary Fig—2 The clustered appearance of the CAAP interactions in the PPI sites is statistically significant (♦♦♦♦♦p < 0.00001). The abundance of the CAAP interactions in the PPI and non-PPI sites was calculated by averaging the % of CAAP interactions from the CAAP alignment samples in Fig. 2 and 3 (Supplementary Table 3). The p value was obtained using a one-way ANOVA.

Supplementary Fig—3 Composition (a) and pairing frequencies (b) of amino acids in the CCAAP boxes from the exemplary 82 crystal structure data. The data for parallel interactions and the antiparallel interactions are shown in dark bars and light bars, respectively. The bar graphs for cysteine, histidine, proline, and tryptophan are not included since they rarely appeared (<32 times).

Supplementary Fig—4 Semi-quantitative assays to test binding affinities. (a) Dot blot analysis to determine the detection limits of the CCAAP-based rAbs, C9-813-92P (monomer) and C9-813-CAA2 (dimer) against the target peptide (PTD12) at various concentrations. (b) Dot blot analysis to compare the detection limits of rAb C9-813-CAA2 (dimer) and the conventional anti-Cas9 Ab-HRP conjugate against the purified Cas9 protein at various concentrations.

Supplementary Fig—5 The CCAAP-based rAb C9-813-CAA2 interacts specifically with the target Cas9 protein in the E. coli BL21 Star (DE3) crude extract. Western blot analysis was carried out using rAb C9-813-CAA2 (dimer) to detect the whole Cas9 protein which is produced in E. coli BL21 Star (DE3) cells harboring pET-Spy-Cas9-dH6. The E. coli BL21 Star (DE3) strain harboring pET-21b was used as negative control.

Supplementary Fig—6 Dot blot analysis to detect the alkaline phosphatase (AP) target sequence (PTD8) using the CCAAP-based oligopeptide synthetic antibodies (sAbs) as 1st Abs: sAb monomer (PTD15) and sAb dimer (PTD16). Synthetic linker-His-tag oligopeptide (PTD20) was used as a negative control sAb. The synthetic oligopeptide PTD7 was used as an unrelated target.

Supplementary Fig—7 Dot blot analysis to detect the PDGF-B target sequence (PTD10) using the CCAAP-based oligopeptide synthetic antibodies (sAbs) as 1st Abs: sAb monomer (PTD17) and sAb dimer (PTD18). Synthetic linker-His-tag oligopeptide (PTD20) was used as a negative control sAb. The synthetic oligopeptide PTD6 was used as an unrelated target.

Supplementary Table—1 Synthetic antibodies (sAbs) and target peptides used in this study.

Supplementary Table—2 Synthetic DNA fragments and oligonucleotides used in this study.

Supplementary Table—3 Appearance of the CAAP interactions in the PPI and non-PPI sites.

Supplementary Table—4 Clustered complementary amino acid pairing (CCAAP) for protein-protein interaction.

Supplementary Table—5 Abundance of the amino acid pairings in the CCAAP boxes from 82 PPI structure data.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (PDF 623 kb)
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Supplementary material 2 (PDF 176 kb)
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Supplementary material 3 (PDF 97 kb)
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Supplementary material 4 (PDF 138 kb)
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Supplementary material 5 (PDF 125 kb)
10529_2018_2616_MOESM6_ESM.pdf (533 kb)
Supplementary material 6 (PDF 533 kb)


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of BioengineeringUniversity of California-BerkeleyBerkeleyUSA
  2. 2.Peption LLCSan DiegoUSA

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