Rational Design of an Antimicrobial Peptide Based on Structural Insight into the Interaction of Pseudomonas aeruginosa Initiation Factor 1 with Its Cognate 30S Ribosomal Subunit

Abstract

Bacterial infections continue to represent a major worldwide health hazard following the emergence of drug-resistant pathogenic strains. Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections with increased morbidity and mortality. The increasing antibiotic resistance in P. aeruginosa has led to an unmet need for discovery of new antibiotic candidates. Bacterial protein synthesis is an essential metabolic process and a validated target for antibiotic development; however, the precise structural mechanism in P. aeruginosa remains unknown. In this work, the interaction of P. aeruginosa initiation factor 1 (IF1) with the 30S ribosomal subunit was studied by NMR, which enabled us to construct a structure of IF1-bound 30S complex. A short α-helix in IF1 was found to be critical for IF1 ribosomal binding and function. A peptide derived from this α-helix was tested and displayed a high ability to inhibit bacterial growth. These results provide a clue for rational design of new antimicrobials.

A rationally designed antimicrobial peptide from structural and functional insights of Clostridioides difficile translation initiation factor 1

“…15 N-labeled Cd-IF1 proteins were expressed and purified as described in the experimental section. The 30S subunits of Pseudomonas aeruginosa ribosomes were prepared according to the procedure reported previously ( 22 ), and used for the titration due to the lack of C. difficile ribosomes. The 1 H- 15 N HSQC NMR spectrum of Cd-IF1 in free form exhibited highly dispersed backbone amide cross peaks, uniform peak intensities, and narrow peak shapes, which were typical characteristics of a well-folded protein.…”

“…The relative intensity/chemical shift changes of two representative amino acids—M21 and H35—were utilized to make an estimate of the apparent dissociation constant (Kd), which is in the micromolar range (Supplementary Materials S2). The binding affinity of Cd-IF1 to the P. aeruginosa 30S ribosomal subunit is somewhat lower than that of Pa-IF1, likely resulting from their structural difference ( 22 ). However, this is consistent with previous studies showing a medium ribosomal binding affinity.…”

A Rationally Designed Antimicrobial Peptide from Structural and Functional Insight ofClostridium difficileTranslation Initiation Factor 1

“…15 N-labeled Cd-IF1 proteins were expressed and purified as described in the experimental section. The 30S subunits of Pseudomonas aeruginosa ribosomes were prepared according to the procedure reported previously [19], and used for the titration due to the lack of C. difficile ribosomes. The 1 H- 15 N heteronuclear single quantum correlation (HSQC) NMR spectrum of Cd-IF1 in free form exhibited highly dispersed backbone amide cross peaks, uniform peak intensities, and narrow peak shapes, which were typical characteristics of a well-folded protein.…”