Conformational studies of a bombolitin III-derived peptide mimicking the four-helix bundle structural motif of proteins

Bombolitins are five structurally related heptadecapeptides originally isolated from the venom of a bumblebee. In aqueous solution, bombolitins at sufficiently high concentration form oligomeric aggregates with consequent conformational transition from a random coil to the α-helical structure. Previous studies suggested that oligomeric aggregates could mimic the four-helix bundle structural motif of proteins. In the present work, we synthesized the following peptide sequence formed by two bombolitin III sequences linked head-to-tail by the tetrapeptide bridge -Gly-Pro-Val-Asp-: I1-K2-I3-M4-D5-I6-L7-A8-K9-L10-G11-K12-V13-L14-A15-H16-V17-G18-P19-V20-D21-I22-K23-I24-M25-D26-I27-L28-A29-K30-L31-G32-K33-V34-L35-A36-H37-V38-NH2. The tetrapeptide GPVD connecting the two helical peptide sequences was chosen to facilitate the formation of the helix−loop−helix structural motif. The conformational properties of the peptide were studied by CD, NMR, and molecular dynamics calculations. The results indicate the presence of a helix−loop−helix conformation at 10-5 M concentration. At higher concentrations, NOESY connectivities were detected which are compatible with the presence of dimers or higher aggregates of peptide molecules in the helix−loop−helix structure packed in an antiparallel fashion. Molecular dynamics simulation were run either with NOE distance restraints or without restraints in explicit solvent for extended time. The results of these simulations support the dimerization of the molecules in the helix−loop−helix structure with formation of the four-helix bundle motif.