Updated publication reference for PubMed record(s): 33199913. We investigated the conformational dynamics of the LPS translocon LptDE was using hydrogen-deuterium exchange mass spectrometry. We evaluated the conformational changes of LptDE upon binding of LPS, Re-LPS (an LPS substructure), thanatin, and the membrane lipid POPG. Our data reveal that LPS induces opening of the LptD β-taco domain, coupled with conformational changes on β-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilises the β-taco, thus blocking these conformational fluctuations and preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both β-barrel and β-taco domains.