Conformational flexibility of EptA driven by an interdomain helix provides insights for enzyme–substrate recognition

Abstract

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.

More than mcr: canonical plasmid- and transposon-encoded mobilized colistin resistance genes represent a subset of phosphoethanolamine transferases

“…The conformational changes of EptA are governed by a highly conserved domain structure that offers extensive flexibility between the membrane-bound and the periplasmic catalytic domains (Anandan et al, 2017;Anandan and Vrielink, 2020a). Moreover, Anandan et al (Anandan et al, 2021) proposed that the bridging helix acts as a hinge region that enables extensive conformational changes between the two domains, "opening up" the catalytic domain to allow access to the considerably large lipid A substrate. Hence, diversification of Ser 191 and Arg 235 is likely to influence the overall conformational flexibility of the enzyme.…”

“…The AA residues predicted to evolve under positive selection were not part of the active site and the substrate-binding site; however, they are likely to affect enzyme function, such as possible alteration of substrate accessibility, specificity, and enzyme efficacy. For example, we predicted that branch-specific positive selection might be involved in the diversification of Ser 191 and Arg 235 in the interdomain-connecting region, which is relevant, as it has been shown that the conformational flexibility of EptA is essential in enzyme-substrate recognition ( Anandan et al., 2021 ). The conformational changes of EptA are governed by a highly conserved domain structure that offers extensive flexibility between the membrane-bound and the periplasmic catalytic domains ( Anandan et al., 2017 ; Anandan and Vrielink, 2020a ).…”

More than mcr: Canonical Plasmid- and Transposon-Encoded Mobilized Colistin Resistance (mcr) Genes Represent a Subset of Phosphoethanolamine Transferases

“…For example, we predicted that branch-specific positive selection might be involved in the diversification of Ser 191 and Arg 235 in the interdomain-connecting region. Recently, it has been shown that the conformational flexibility of EptA is essential in enzyme-substrate recognition (Anandan et al, 2021). The conformational changes of EptA are governed by a highly conserved domain structure that offers extensive flexibility between the membrane-bound and the periplasmic catalytic domains (Anandan et al, 2017; Anandan and Vrielink, 2020).…”

“…The conformational changes of EptA are governed by a highly conserved domain structure that offers extensive flexibility between the membrane-bound and the periplasmic catalytic domains (Anandan et al, 2017; Anandan and Vrielink, 2020). Moreover, Anandan et al (Anandan et al, 2021) proposed that the bridging helix acts as a hinge region that enables extensive conformational changes between the two domains, “opening up” the catalytic domain to allow access to the considerably large lipid A substrate. Hence, diversification of Ser 191 and Arg 235 is likely to influence the overall conformational flexibility of the enzyme.…”

Boosting the Full Potential of PyMOL with Structural Biology Plugins

“…The implementation in the PyMOL framework makes it accessible for regular use, even by non-experts. For instance, MPBuilder was used for the interpretation of SAXS data of the phosphoethanolamine enzyme that is active at the lipidic interface, namely lipid A of pathogenic gram-negative bacteria as an antibiotic resistance mechanism [ 55 ].…”