Expanding the paradigm of glycopeptide antibiotic recognition through molecular dynamics simulations

Glycopeptide antibiotics (GPAs) are important therapeutics that canonically bind the D-Ala-D-Ala terminus of peptidoglycan precursors. However, recent work suggests they can also engage other targets within the bacterial cell wall. Keratinicyclin B (KCB), for example, is believed to bind the wall teichoic acid, polysaccharide II (PSII). Although GPA–peptidoglycan interactions are well studied, the molecular determinants of KCB specificity remain unclear. Herein, we employ molecular dynamics simulations and continuum solvent free-energy analysis to probe how scaffold variations, glycosylation patterns, and functional group modifications govern GPA recognition of canonical (peptidoglycan) and noncanonical (PSII) ligands. Our results demonstrate that features beyond the characteristic hydrogen bonding network, including steric preorganization, electrostatics, and binding site solvation, collectively shape the energetic landscape. Simulations of KCB–PSII and in silico variants reveal a layered recognition strategy, highlighting both essential and compensatory contributions to target binding. Notably, upon disruption of the primary binding interface, PSII adopts alternative poses stabilized by a secondary interface with GPA glycosyl groups. Together, these findings expand the paradigm of GPA recognition and provide a mechanistic framework for designing next-generation antibiotics.