Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)

This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the beta(2)-adrenergic receptor (beta(2)-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of beta(2)-AR were analysed using the informational spectrum method (ISM) and beta(2)-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the beta(2)-AR:P3 complex of Delta G= (-7.23 +/- 0.04) kcal/mol, or a Kd of (7.9 +/- 0.5) mu M, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated beta(2)-AR, which displayed similar to 10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 mu M vs. 58.22 mu M), while its ability to inhibit the agonist-induced interaction of beta(2)-AR with beta-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated beta(2)-AR.