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Identifying key juxtamembrane interactions in cell membranes using AraC-based transcriptional reporter assay (AraTM).

ABSTRACT: Dimerization is a key regulatory mechanism in activation of transmembrane (TM) receptors during signal transduction. This process involves a coordinated interplay between extracellular (EX), TM, and cytoplasmic (CYTO) regions to form a specific interface required for both ligand binding and intracellular signaling to occur. While several transcriptional activator-based methods exist for investigating TM interactions in bacterial membranes, expression of TM chimera in these methods occurs in a reverse orientation, and are limited to only TM domains for proper membrane trafficking and integration. We therefore developed a new, AraC-based transcriptional reporter assay (AraTM) that expresses EX-TM-CYTO chimera in their native orientation, thereby enabling membrane trafficking to occur independent of the TM chimera used as well as permitting analysis of EX-TM-CYTO interactions in biological membranes. Using integrin α(IIb) TM-CYTO as a model, we observe a large increase in homodimerization for the constitutively active TM mutant L980A relative to wild-type in the TM-CYTO construct (A963-E1008). We also characterized the receptor for advanced glycation endproducts (RAGE), whose homooligomeric state is critical in ligand recognition, and find the specific juxtamembrane region within the CYTO (A375-P394) mediates homodimerization, and is dominant over effects observed when the extracellular C2 domain is included. Furthermore, we find good agreement between our AraTM measurements in bacterial membranes and BRET measurements made on corresponding RAGE constructs expressed in transfected HEK293 cells. Overall, the AraTM assay provides a new approach to identify specific interactions between receptor EX-TM-CYTO domains in biological membranes that are important in regulation of signal transduction.

SUBMITTER: Su PC

PROVIDER: S-EPMC3438984 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Teixobactin (Txb) is a recently discovered antibiotic against Gram-positive bacteria that induces no detectable resistance. The bactericidal mechanism is believed to be the inhibition of cell wall biosynthesis by Txb binding to lipid II and lipid III. Txb binding specificity likely arises from targeting of the shared lipid component, the pyrophosphate moiety. Despite synthesis and functional assessment of numerous chemical analogs of Txb, and consequent identification of the Txb pharmacophore, the detailed structural information of Txb-substrate binding is still lacking. Here, we use molecular modeling and microsecond-scale molecular dynamics simulations to capture the formation of Txb-lipid II complexes at a membrane surface. Two dominant binding conformations were observed, both showing characteristic lipid II phosphate binding by the Txb backbone amides near the C-terminal cyclodepsipeptide (d-Thr8-Ile11) ring. Additionally, binding by Txb also involved the side chain hydroxyl group of Ser7, as well as a secondary phosphate binding provided by the side chain of l-allo-enduracididine. Interestingly, those conformations differ by swapping two groups of hydrogen bond donors that coordinate the two phosphate moieties of lipid II, resulting in opposite orientations of lipid II binding. In addition, residues d-allo-Ile5 and Ile6 serve as the membrane anchors in both Txb conformations, regardless of the detailed phosphate binding interactions near the cyclodepsipeptide ring. The role of hydrophobic residues in Txb activity is primarily for its membrane insertion, and subsidiarily to provide non-polar interactions with the lipid II tail. Based on the Txb-lipid II interactions captured in their complexes, as well as their partitioning depths into the membrane, we propose that the bactericidal mechanism of Txb is to arrest cell wall synthesis by selectively inhibiting the transglycosylation of peptidoglycan, while possibly leaving the transpeptidation step unaffected. The observed "pyrophosphate caging" mechanism of lipid II inhibition appears to be similar to some lantibiotics, but different from that of vancomycin or bacitracin.

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Identifying key juxtamembrane interactions in cell membranes using AraC-based transcriptional reporter assay (AraTM).

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