Exploration of the Sequence of Antimicrobial Peptide Api-137, a Translation Termination Inhibitor

Proline-rich antimicrobial peptides (PrAMPs) are endogenously expressed by multicellular organisms as a part of their innate immune response. These PrAMPs have been identified as a source of novel antibiotics. Previous studies indicate that Apidaecin, a PrAMP produced by European honeybees, passively permeates the outer membrane of Gram-negative bacteria and reaches the cytoplasm via the SbmA transporter located in the inner membrane. In the cytoplasm, Apidaecin inhibits translation termination by binding in the nascent peptide exit tunnel after peptidyl-tRNA hydrolysis and trapping release factors on the ribosome. Structural analysis shows that the Apidaecin C-terminus interacts with the P-site t-RNA and the A-site bound release factor, while the N-terminus stretches down the length of the exit tunnel. This orientation mimics that of the nascent peptide, therefore, multiple Apidaecin residues interact with rRNA nucleotides and ribosomal proteins of the exit tunnel; however, it is unclear which of these interactions are necessary for binding and inhibitory action. Informed by structural and biochemical data, synthetic Apidaecin variants were designed, synthesized, and tested to determine necessary binding interactions and to improve its antimicrobial activity and proteolytic stability. Unnatural amino acids and constraints were incorporated into the Apidaecin sequence. This work provides compelling new research avenues for a novel antibiotic mechanism.