The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release

Thomas E. Bohl, Pek Ieong, John K. Lee, Thomas Lee, Jayakanth Kankanala, Ke Shi, Özlem Demir, Kayo Kurahashi, Rommie E. Amaro, Zhengqiang Wang, Hideki Aihara

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen– deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.

Original languageEnglish (US)
Pages (from-to)7969-7981
Number of pages13
JournalJournal of Biological Chemistry
Volume293
Issue number21
DOIs
StatePublished - May 25 2018

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health Grants GM118047 (to H. A.) and DP2-OD007237, NIGMS P41-GM103426 and CHE060073N (to R. E. A.). J. K. L. is currently employed by Bristol-Myers Squibb (Redwood City, CA). J. K. L. was not employed by BMS during involvement in this research. R. E. A. is a co-founder of, on the scientific advisory board of, and has equity interest in Actavlon, Inc. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen– deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.

Funding Information:
This work was supported by National Institutes of Health Grants GM118047 (to H. A.) and DP2-OD007237, NIGMS P41-GM103426 and CHE060073N (to R. E. A.). J. K. L. is currently employed by Bristol-Myers Squibb (Redwood City, CA). J. K. L. was not employed by BMS during involvement in this research. R. E. A. is a co-founder of, on the scientific advisory board of, and has equity interest in Actavlon, Inc. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Surajit Banerjee for assistance at the Advanced Photon Source. In addition, we thank Kurt Peterson of Fluorescence Innovations for help in measuring luminescence. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are supported by NIGMS, National Institutes of Health Grant P41 GM103403. The Eiger 16M detector on 24-ID-E Beamline is funded by National Institutes of Health-ORIP HEI Grant S10OD021527. This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. UCSF Chimera was utilized for protein structure analysis and to generate figures; Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, which is supported by NIGMS, National Institutes of Health Grant P41-GM103311.

Publisher Copyright:
© 2018 Bohl et al.

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