Conformational Snapshots of CydDC in a Native Lipid Bilayer Coupling Heme Transport to Antibiotic Resistance

ABSTRACT Heme is an essential cofactor in numerous biological processes, including bacterial respiration. The ABC transporter CydDC facilitates the assembly and maturation of the cytochrome bd terminal oxidase by exporting heme and has been implicated in antibiotic resistance in bacteria. However, the dynamic conformations of CydDC in a native‐like lipid bilayer remain unresolved, and its resistance mechanism is still elusive. Here, we determined high‐resolution cryo‐electron microscopy structures of nanodisc‐reconstituted CydDC in the apo, nucleotide‐bound and heme‐bound states, providing direct structural evidence for its substrate‐stimulated hydrolysis mechanism. In vivo and in vitro biochemical characterization identified key residues of CydDC that are critical for substrate binding and transport. Bioinformatics analysis further demonstrated that CydDC is highly conserved across bacterial species. Transcriptomic profiling of cydC/D in antibiotic‐resistant strains showed that elevated expression of cydC/D correlates with increased antibiotic resistance. Moreover, mutations at the heme‐binding sites altered bacterial susceptibility to multiple antibiotics, suggesting that the exporting activity of CydDC may also contribute directly to drug resistance. Together, these findings provide mechanistic insights into CydDC‐mediated heme transport and potential drug efflux, and inform the development of antimicrobial strategies targeting the respiratory chain.