Local structural constraints and charge distribution in the protegrin-1 arginine loop govern activity against colistin-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae infections are increasingly treated with colistin as a last-resort antibiotic, but colistin resistance is rising, largely due to PhoPQ-mediated lipid A modifications. Host defense peptides (HDPs) and colistin are both cationic and amphipathic, yet HDPs are not uniformly affected by lipid A remodeling. To understand how certain HDPs can penetrate colistin-resistant membranes, we performed deep mutational scanning of the β-hairpin peptide protegrin-1 against colistin-resistant K. pneumoniae MKP103. We focused on the triple arginine loop region and systematically probed single and double mutants using surface display, MIC assays, membrane permeabilization analyses, and molecular modeling. A single arginine-to-proline substitution at position 9, within the β-sheet-to-turn transition, reduced activity despite preserved disulfide bonds, whereas an analogous substitution at position 11, in the turn region, did not. Double mutations that removed two arginine amino acids in this loop abolished activity and were associated with decreased inner membrane leakage and modeled pores of reduced diameter. Molecular dynamics simulations showed that disruptive mutations in the β-sheet-to-turn region accelerated loss of β-sheet character and facilitated anti-parallel dimer orientation in the membrane. Together, these findings demonstrate that subtle, position-specific structural perturbations in the arginine loop can have equal impact as changes in net positive charge in determining protegrin-1 activity against colistin-resistant K. pneumoniae.