Structural basis for EtfD-mediated coupling of β-oxidation and the respiratory chain in mycobacteria

Targeting β-oxidation has been proposed as a strategy for shortening tuberculosis (TB) treatment by killing non-replicating Mycobacterium tuberculosis within granulomas where the pathogen relies on host-derived lipids. The protein EtfD is thought to couple β-oxidation of fatty acids with the respiratory chain in mycobacteria. However, the structure of EtfD is not known and, as the presumed link between two complex processes, its activity has been difficult to measure, impeding its exploitation as a drug target. Here we show that Mycobacterium smegmatis, a fast growing and nonpathogenic model for M. tuberculosis, relies on EtfD for extracting energy from β-oxidation. The electron cryomicroscopy structure of M. smegmatis EtfD reveals an unusual linear [3Fe-4S] cluster that has not been seen in other protein structures, and suggests how EtfD transfers electrons from β-oxidation to the respiratory chain. We devised an assay that couples EtfD activity to a fluorescent readout of proton pumping by the respiratory chain, which can be used to identify compounds that block mycobacteria from using β-oxidation to power oxidative phosphorylation.