TY - JOUR
T1 - Mechanism underlying autoinducer recognition in the Vibrio cholerae DPO-VqmA quorum-sensing pathway
AU - Huang, Xiuliang
AU - Duddy, Olivia P.
AU - Silpe, Justin E.
AU - Paczkowski, Jon E.
AU - Cong, Jianping
AU - Henke, Brad R.
AU - Bassler, Bonnie L.
N1 - Funding Information:
Acknowledgments—We thank Dr. Phillip Jeffrey of the Macromolecular Crystallography Core Facility, Dr. Saw Kyin of the Proteomics and Mass Spectrometry Core Facility, and Dr. Venu Vandasvasi of the Biophysics Core Facility, all at Princeton University, for expert assistance. We thank Dr. Frederick Hughson for thoughtful discussions. We thank the Bassler group for insight into the research. This research used resources FMX and AMX of the National Synchrotron Light Source II, a United States Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract DE-SC0012704. The Life Science Biomedical Technology Research resource is primarily supported by NIGMS, National Institutes of Health, through Biomedical Technology Research Resource P41 Grant P41GM111244 and by DOE Office of Biological and Environmental Research Grant KP1605010. The authors received expert technical support from Dr. Martin Fuchs and Dr. Jean Jakoncic of the NSLS II FMX and AMX beamlines.
Funding Information:
This work was supported by the Howard Hughes Medical Institute, National Institutes of Health Grant 5R37GM065859, and National Science Foundation Grant MCB-1713731 (to B. L. B.); a Jane Coffin Childs Memorial Fund for Biomedical Research Postdoctoral Fellowship (to J. E. P.); NIGMS, National Institutes of Health, Grant T32GM007388 (to O. P. D.); and a Charlotte Elizabeth Procter Fellowship provided by Princeton University and a National Defense Science and Engineering Graduate Fellowship supported by the Department of Defense (to J. E. S.). The authors declare that they have no conflicts of interest with the contents of this article. 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:
This work was supported by the Howard Hughes Medical Institute, National Institutes of Health Grant 5R37GM065859, and National Science Founda-tion Grant MCB-1713731 (to B. L. B.); a Jane Coffin Childs Memorial Fund for Biomedical Research Postdoctoral Fellowship (to J. E. P.); NIGMS, National Institutes of Health, Grant T32GM007388 (to O. P. D.); and a Charlotte Elizabeth Procter Fellowship provided by Princeton Univer-sity and a National Defense Science and Engineering Graduate Fellow-ship supported by the Department of Defense (to J. E. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 Huang et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/3/6
Y1 - 2020/3/6
N2 - Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-di-methyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2'-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.
AB - Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-di-methyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2'-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.
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U2 - 10.1074/jbc.RA119.012104
DO - 10.1074/jbc.RA119.012104
M3 - Article
C2 - 31964715
AN - SCOPUS:85081132049
SN - 0021-9258
VL - 295
SP - 2916
EP - 2931
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -