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. 2024 Jan 5:10:1332359.
doi: 10.3389/fmolb.2023.1332359. eCollection 2023.

Design of protein-binding peptides with controlled binding affinity: the case of SARS-CoV-2 receptor binding domain and angiotensin-converting enzyme 2 derived peptides

Giacomo Parisi #  1 , Roberta Piacentini #  2 , Alessio Incocciati  2 , Alessandra Bonamore  2 , Alberto Macone  2 , Jakob Rupert  3   4 , Elsa Zacco  4 , Mattia Miotto  5 , Edoardo Milanetti  6   5 , Gian Gaetano Tartaglia  3   4 , Giancarlo Ruocco  5   6 , Alberto Boffi  2 , Lorenzo Di Rienzo  5
Affiliations

Design of protein-binding peptides with controlled binding affinity: the case of SARS-CoV-2 receptor binding domain and angiotensin-converting enzyme 2 derived peptides

Giacomo Parisi et al. Front Mol Biosci. .

Abstract

The development of methods able to modulate the binding affinity between proteins and peptides is of paramount biotechnological interest in view of a vast range of applications that imply designed polypeptides capable to impair or favour Protein-Protein Interactions. Here, we applied a peptide design algorithm based on shape complementarity optimization and electrostatic compatibility and provided the first experimental in vitro proof of the efficacy of the design algorithm. Focusing on the interaction between the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor, we extracted a 23-residues long peptide that structurally mimics the major interacting portion of the ACE2 receptor and designed in silico five mutants of such a peptide with a modulated affinity. Remarkably, experimental KD measurements, conducted using biolayer interferometry, matched the in silico predictions. Moreover, we investigated the molecular determinants that govern the variation in binding affinity through molecular dynamics simulation, by identifying the mechanisms driving the different values of binding affinity at a single residue level. Finally, the peptide sequence with the highest affinity, in comparison with the wild type peptide, was expressed as a fusion protein with human H ferritin (HFt) 24-mer. Solution measurements performed on the latter constructs confirmed that peptides still exhibited the expected trend, thereby enhancing their efficacy in RBD binding. Altogether, these results indicate the high potentiality of this general method in developing potent high-affinity vectors for hindering/enhancing protein-protein associations.

Keywords: biolayer interferometry (BLI); ferritin-based nanoparticle; molecular dynamics simulation; peptide design; shape complementarity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

FIGURE 1
FIGURE 1
Crystal structure of SARS-CoV-2 spike receptor-binding domain bound with ACE2 (PDB id: 6VW1). Relevant side chains of the residues in the α-helix are shown in the orange box.
FIGURE 2
FIGURE 2
Analysis of the peptides molecular dynamics. (A) Root Mean Square Deviation (RMSD) as a function of time with respect to the initial configuration for each of the six peptides. (B) Density distribution of the spatial distance between the terminal α-carbon, for all of six peptides simulations.
FIGURE 3
FIGURE 3
Time-course of the reaction between peptides and RBD as the analyte protein in solution at various concentrations. For all measurements, a 300 s time period was set for both association and dissociation steps. Peptides have been loaded first at fixed concentration (50 μg/mL) on biosensors initialized with streptavidin molecule.
FIGURE 4
FIGURE 4
Peptide-spike complexes molecular dynamics. (A) Boxplots describing the percentage of conserved contacts at the equilibrium, with respect to those observed in the starting structure. (B) The average contacts each peptide residue made during the equilibrium simulation frames. Each color represents a specific mutant peptide-Spike complex. (C) Intermolecular links characterized by an occurrence difference higher than 50% with respect to the wild type. (D) Molecular representation of the Peptide-Spike complex with highlighted the 30:455 and 41:501 links, acquired by the high affinity mutants. (E) Molecular representation of the Peptide-Spike complex with highlighted the 34:417 and 41:500 links, lost by the high affinity mutants.
FIGURE 5
FIGURE 5
Biochemical characterization of ACE2WT-HFt and ACE2HA1-HFt compared to HFt. (A) HP-SEC analysis. (B) SDS-PAGE (lane 1: marker, lane 2: HFt, lane3: ACE2WT-HFt, lane 4: ACE2HA1-HFt). (C) native gel electrophoresis (lane1: HFt, lane2: ACE2WT-HFt, lane 3: ACE2HA1-HFt).
FIGURE 6
FIGURE 6
Time courses of the reaction between RBD and ferritin constructs. RBD protein has been loaded first at fixed concentration (50 μg/mL) on Ni-NTA biosensors. (A) ferritin with WT peptide construct is the analyte protein in solution at various concentrations. We set a 70 s time interval for the association step and 80 s for dissociation. (B) ferritin with HA1 peptide construct is the analyte protein in solution at various concentrations. Association and dissociation steps have duration respectively of 70 s and 80 s. Temperature was 25 °C.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was partially funded by grants from ERC-2019-Synergy Grant (ASTRA, no. 855923); EIC-2022-PathfinderOpen (ivBM-4PAP, no. 101098989); Project “National Center for Gene Therapy and Drugs based on RNA Technology” (CN00000041) financed by Next-Generation EU PNRR MUR—M4C2—Action 1.4—Call “Potenziamento strutture di ricerca e creazione di “campioni nazionali di R&S” (CUP J33C22001130001). Moreover, this research was partially funded by PAN-HUB project no T4-AN-07, “Traiettoria 4 del Piano Sviluppo e Coesione Salute”—FSC 2014-2020, to ABof.