Conformational flexibility of EptA driven by an interdomain helix provides insights for enzyme–substrate recognition
Anandhi Anandan,
Nicholas W. Dunstan,
Timothy M. Ryan,
Haydyn D. T. Mertens,
Katherine Y. L. Lim,
Genevieve L. Evans,
Charlene M. Kahler,
Alice Vrielink
Affiliations
Anandhi Anandan
School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Perth 6009, Australia
Nicholas W. Dunstan
School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Perth 6009, Australia
Timothy M. Ryan
Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia
Haydyn D. T. Mertens
European Molecular Biology Laboratory, Hamburg Unit, DESY, Notkestrasse 85, 22607 Hamburg, Germany
Katherine Y. L. Lim
The Marshall Centre for Infectious Diseases Research and Training, School of Biomedical Sciences, University of Western Australia, 35 Stirling Highway, Perth, Western Australia 6009, Australia
Genevieve L. Evans
School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Perth 6009, Australia
Charlene M. Kahler
The Marshall Centre for Infectious Diseases Research and Training, School of Biomedical Sciences, University of Western Australia, 35 Stirling Highway, Perth, Western Australia 6009, Australia
Alice Vrielink
School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Perth 6009, Australia
Many pathogenic gram-negative bacteria have developed mechanisms to increase resistance to cationic antimicrobial peptides by modifying the lipid A moiety. One modification is the addition of phosphoethanolamine to lipid A by the enzyme phosphoethanolamine transferase (EptA). Previously we reported the structure of EptA from Neisseria, revealing a two-domain architecture consisting of a periplasmic facing soluble domain and a transmembrane domain, linked together by a bridging helix. Here, the conformational flexibility of EptA in different detergent environments is probed by solution scattering and intrinsic fluorescence-quenching studies. The solution scattering studies reveal the enzyme in a more compact state with the two domains positioned close together in an n-dodecyl-β-d-maltoside micelle environment and an open extended structure in an n-dodecyl-phosphocholine micelle environment. Intrinsic fluorescence quenching studies localize the domain movements to the bridging helix. These results provide important insights into substrate binding and the molecular mechanism of endotoxin modification by EptA.
