Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells

Abstract

Monitoring the assembly of macromolecules to design entities with novel properties can be achieved either chemically creating covalent bonds or by non-covalent connections using appropriate structural motifs. In this report, two self-associating peptides (named K3 and E3) that originate from p53 tetramerization domain were developed as tools for highly specific and non-covalent heterotetramerization of two bio-molecules. The pairing/coupling preferences of K3 and E3 were first evaluated by molecular modeling data and confirmed using circular dichroism spectroscopy, size-exclusion chromatography and biological assays. Regardless of the moieties fused to K3 and E3, these two peptides self-assembled into dimers of dimers to form bivalent heterotetrameric complexes that proved to be extremely stable inside of living cells. The benefits of the multivalency in terms of avidity, specificity and expanded functional activity were strikingly revealed when the Proliferating Cell Nuclear Antigen (PCNA), which is essential for DNA replication, was targeted using a heterotetramer presenting both an antibody fragment against PCNA and a specific PCNA binder peptide. In vitro heterotetramerization of these two known PCNA ligands increased their binding efficiencies to PCNA up to 80-fold compared to the best homotetramer counterpart. In cellulo, the heterotetramers were able to efficiently inhibit DNA replication and to trigger cell death. Altogether, we demonstrate that these two bi-selective self-assembling peptidic domains offer a versatile non-covalent conjugation method that can be easily implemented for protein engineering.

Small p53 derived peptide suitable for robust nanobodies dimerization

“…We use the well-characterized nanobody directed against the green fluorescent protein, eGFP, (cAbGFP4) referred herein as nano-eGFP-mono that has been described to have a K D measured by SPR of 0.32 nM (Saerens et al, 2005). Bivalency was achieved either by genetically fusing the nano-eGFP coding sequence in a tandem with a spacer composed of four glycine residues followed by one serine repeated 3 times (hereinafter referred as to nano-eGFP-tandem), or via the self-associating peptide E3 (hereinafter refereed as to nano-eGFP-E3), which originates from the tetramerization domain of p53 (residues 325-355) (Figure 1A) (Vigneron et al, 2019). The paratopes of these two bivalent nanobodies have a different orientation relatively to each other, namely a tail-to-head and a tail-to-tail orientation, respectively.…”

“…Cells were transiently transfected with expression plasmids using JetpEI reagent (Polyplus transfection, Illkirch) according to the manufacturer's protocol or electro-transfected with proteins as previously described (Freund et al, 2013). elsewhere (Baldeck et al, 2015;Freund et al, 2014;Masson et al, 2003;Shima et al, 1997;Vigneron et al, 2019). The doubled-stranded DNA sequence oligonucleotide encoding a mitochondriatargeting signal (MTS) MISLTARLSRSAPDSQACYSV-LGAFLGKCADV was cloned between the HindIII and the PstI sites of pEGFP-N1 vector.…”

“…We have recently taken advantage of a modified tetramerization domain of p53 to construct bifunctional and bispecific heterotetramer biomolecules (Stoessel et al, 2020;Vigneron et al, 2019). This tetramerization domain (p53tet) is a small peptide of 31-aminoacids, which can be fused to various moieties.…”

Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides

“…In this study, we developed a straightforward co-assembly technique to produce an immunotoxin combining a nanobody directed against HER2 and a toxin via two peptides E3 and K3. 16 The non-covalent and highly specific pairing of the E3 and K3 peptides yielded to the generation of heterotetrameric immunotoxins, composed of two VHH and two toxin molecules. We clearly demonstrate that the engineered immunotoxins retain their specific binding and toxic properties, hence being specifically toxic for HER2-overexpressing breast cancer cells.…”

“…HER2 PCR fragment was digested with NcoI-SpeI and ligated into the pETOM-P40M1-E3 or -K3 digested with NcoI-SpeI to replace the P40M1 sequences. 16 An optimized sequence encoding for the TOX used in this study was synthetized by Integrated DNA Technologies (IDT) and amplified by PCR using specific primers:…”