Chemoproteomics reveals disruption of metal homeostasis and metalloproteins by the antibiotic holomycin

The natural product holomycin contains a unique cyclic disulfide and exhibits broad-spectrum antimicrobial activities. Reduced holomycin chelates metal ions with high affinity and disrupts metal homeostasis in the cell. To identify cellular metalloproteins that are affected by holomycin, reactive-cysteine profiling was performed using isotopic Tandem Orthogonal Proteolysis–Activity-based Protein Profiling. This chemoproteomic analysis showed that holomycin treatment increases the reactivity of metal-coordinating cysteine residues in several zinc-dependent and iron-sulfur cluster-dependent enzymes. Whole-proteome abundance analysis revealed that holomycin treatment induces zinc starvation, iron starvation, and cellular stress. This study sets the stage for investigating the impact of metal-binding molecules on metalloproteomes using chemoproteomics.