Glycopeptide antibiotics, GPAs, have been cornerstones of gram-positive infection therapy since vancomycin entered clinical use in 1958. Their canonical mechanism centers on five hydrogen bonds that anchor the D-Ala-D-Ala terminus of the peptidoglycan precursor lipid II within a preorganized binding cleft, sterically blocking both transglycosylation and transpeptidation. Decades of structural and biochemical work have mapped this interaction in fine detail. Yet the recent discovery of keratinicyclin B, KCB, a GPA isolated from Amycolatopsis keratiniphila that targets a species-specific wall teichoic acid polysaccharide rather than the peptidoglycan terminus, signals that the canonical hydrogen-bonding picture captures only part of the recognition story.
Researchers in the Davis Group at Emory University, published in Communications Chemistry, conducted molecular dynamics, MD, simulations of vancomycin, keratinimicin A, KMA, and KCB in complex with their respective cell wall targets, then extended the analysis to a panel of in silico KCB variants to dissect the contribution of individual functional groups. Triplicate 100 ns trajectories were evaluated by root-mean-square-deviation analysis and molecular mechanics Poisson–Boltzmann surface area, MM-PBSA, free-energy calculations. Starting models were geometry-optimized by DFT, and select trajectories were extended to 300 ns to assess the stability of alternative binding modes.
Simulations of the three GPAs with a diacetyl-L-Lys-D-Ala-D-Ala tripeptide recapitulated experimentally measured affinities. Vancomycin and KMA each formed stable complexes, with average tripeptide backbone RMSDs of 1.5 ± 0.6 Å and 1.0 ± 0.4 Å, respectively. KCB dissociated from the tripeptide despite retaining accessible hydrogen-bonding partners, reaching an RMSD of 5.5 ± 2.7 Å, consistent with a measured dissociation constant roughly 100-fold weaker than vancomycin. Comparison with desleucyl vancomycin, dVAN, a truncated hexapeptide that binds the D-Ala-D-Ala motif even more weakly, clarified the mechanism: the researchers propose that reduced tripeptide binding by KCB primarily reflects the absence of steric capping by the seventh residue, while the exceptionally low affinity of dVAN arises from additional electrostatic repulsion between the tripeptide carboxylate and the N-terminal backbone carbonyl of the hexapeptide scaffold. MM-PBSA analysis further showed that binding-site desolvation imposes an entropic penalty large enough that apparently favorable computed free energies for unstable complexes reflect the favorability of dissociation rather than stable complex formation.
Simulations of the KCB–polysaccharide II, PSII, complex revealed a distinct recognition logic. The PSII phosphodiester group proved dominant: removing it produced RMSD values of 10.3 ± 3.1 Å and complete loss of canonical binding-site engagement, matching in vitro data. Replacing the oxazolidinone N1 or carbonyl oxygen with carbon produced similarly large displacements, with RMSDs of 9.2 ± 3.8 Å and 10.6 ± 3.0 Å respectively, identifying these atoms as critical anchors. By contrast, modifications to the βGalNAc4 N-acetyl group or the L-Phe3 backbone amide produced moderate perturbations that the complex accommodated through compensatory contacts. In the L-Phe3 variant, PSII populated two distinct conformations: one in which βGalNAc4 migrated toward a secondary interface formed by the GPA glycosyl groups, and one in which it remained near the primary site through a new contact with the D-Hpg4 backbone. Extended 300 ns trajectories of the oxazolidinone carbonyl variant confirmed that once primary-site capture is disrupted, the contracted secondary-binding conformation is stably maintained through a salt bridge between the PSII phosphate and the cationic amine of the acosamine sugar, with additional support from an extensive hydrogen-bonding network involving neighboring PSII sugars.
These findings reframe GPA recognition as a multilayered process in which steric preorganization, electrostatic complementarity, and binding-site solvation jointly shape complex stability alongside hydrogen bonding. The secondary binding site observed in KCB simulations parallels a mode proposed for desleucyl-oritavancin, where alternative PG precursor poses engage the pentaglycine bridge via hydrophobic contacts; KCB achieves an analogous fallback via electrostatic interactions with polar GPA sugars instead. The residue at position 3 of the heptapeptide scaffold also emerges as a conserved intramolecular surrogate that occludes the binding cleft when no ligand is present, preserving site accessibility across chemotypes. Together, the results provide a mechanistic framework for designing next-generation GPAs that leverage multiple binding modes to broaden therapeutic potential against resistant pathogens including Clostridioides difficile.
