Molecular Design of Sequence‐minimized, Structure‐optimized, and Hydrocarbon‐stapled Helix–Helix Interactions in the Trimer‐of‐Hairpins Motif of Pediatric Pneumonia RSV‐F Protein

The minimum unit of helix–helix interaction is stripped from the six‐helix bundle of RSV‐F trimer‐of‐hairpins motif, which is then truncated to obtain a core binding region (CBR) consisting of a spontaneously structured HRn peptide and an intrinsically disordered HRc peptide. Residue mutagenesis and hydrocarbon stapling are rationally used to constrain the conformational rigidity and helical propensity of HRc, thus considerably improving its binding affinity to HRn.

Abstract

The respiratory syncytial virus fusion (RSV‐F) protein is a primary target for vaccine and drug development against respiratory infection and pediatric pneumonia. The RSV‐F core forms a trimer‐of‐hairpins (TOH) motif in post‐fusion conformation, which is characterized by a six‐helix bundle (6HB) where the three N‐terminal HRn helices define a central coiled‐coil, while three C‐terminal HRc helices pack on the coiled‐coil surface in an antiparallel manner. Here, one tightly packed HRn–HRc helix–helix interaction is stripped from the 6HB, which represents the minimum unit of RSV‐F TOH motif. The helix–helix interaction sequence can be truncated to derive a core binding region (CBR) that covers intense nonbonded interactions across the interaction interface. Dynamics simulation and energetics analysis reveal that the CBR HRc peptide has a large flexibility and intrinsic disorder in unbound free state, which would incur a considerable entropy penalty upon its binding to CBR NRn peptide. Two strategies are described to constrain the HRc peptide conformation. First, the four non‐interfacial residues of HRc peptide are artificially substituted with new amino acid combinations of high helical propensity and, second, the helical conformation of wild‐type and mutant HRc peptides is stabilized by adding an all‐hydrocarbon bridge across two spatially vicinal, non‐interfacial residues 503 (i) and 507 (i+4). Free energy calculation and fluorescence‐based assay confirm that the substitution and stapling can effectively improve the binding affinity of CBR HRn–HRc interaction.

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