Charting the sequence-activity landscape of peptide inhibitors of translation termination

Abstract

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api’s activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

A platform for deep sequence-activity mapping and engineering antimicrobial peptides

“…Non-leaky, inducible control of AMP expression has been achieved in pBAD systems in E. coli , not requiring a T7 RNA polymerase. 23, 29 Similarly, extension to AMPs beyond oncocin is conceptually straightforward with the current limitation of requiring an intracellular target, which encompasses a broad swath of AMPs.…”

“…coarsely screened 336 apidaecin AMP mutants with a similar approach. 23 We present an enhanced approach to screen over 10 5 AMP variants simultaneously, uncover sequence determinants of antimicrobial activity, and leverage the sequence-activity landscape to engineer AMPs with enhanced activity.…”

The Multifaceted Roles of Proline in Cell Behavior

“…L-Proline-rich antimicrobial peptides (PrAMPs), involved in innate immunity, are the first line of defense against infections (Graf and Wilson, 2019), and they contain up to 50% L-Pro residues, and are secreted by insects, crustaceans and mammals (Table 3; Mishra et al, 2018). Mechanistically, PrAMPs are channeled by the peptide antibiotic transporter SbmA into the bacterial cytoplasm (Mattiuzzo et al, 2007;Runti et al, 2013), where they bind ribosomal proteins and inhibit protein synthesis (Figure 2 and Table 3; Graf et al, 2017;Graf and Wilson, 2019;Baliga et al, 2021).…”

Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

“…Apidaecin uptake requires the sbmA gene, which encodes an inner-membrane transport protein (32); sbmA is missing in some Gram-negative and all Gram-positive bacterial species. If apidaecin uptake is low, an alternative approach would be to encode and express apidaecin, a small protein, inside the bacteria (33,34). Similarly, Onc112, which functions similarly to retapamulin, is a small protein that could be encoded and expressed within cells (35).…”