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498 Structural Determination of the CqsR CACHE Domain and its Autoinducer

Part of: JCTS_2023_ABSTRACT_COLLECTION

Published online by Cambridge University Press:  24 April 2023

Andrew Guarnaccia ,
Wei-Lung Ng ,
Anjali Steenhaut ,
Sandra Olenic ,
Lark J. Perez  and
Matthew B. Neiditch
Andrew Guarnaccia
Affiliation:
Department of Microbiology, Biochemistry, and Molecular Genetics, New Jersey, Medical School, Rutgers
Wei-Lung Ng
Affiliation:
Department of Molecular Biology and Microbiology, Tufts University School of Medicine
Anjali Steenhaut
Affiliation:
Department of Molecular Biology and Microbiology, Tufts University School of Medicine
Sandra Olenic
Affiliation:
Department of Molecular Biology and Microbiology, Tufts University School of Medicine
Lark J. Perez
Affiliation:
Department of Chemistry & Biochemistry, Rowan University
Matthew B. Neiditch
Affiliation:
Department of Microbiology, Biochemistry, and Molecular Genetics, New Jersey, Medical School, Rutgers
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Abstract

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OBJECTIVES/GOALS: Our goal is to determine the structure of the CACHE domain of the Vibrio cholerae quorum sensing receptor CqsR as well as its autoinducer (AI). We are performing X-ray crystallography on the protein in its apo form, with the fractions containing the AI, and with known ligand ethanolamine (ETA). METHODS/STUDY POPULATION: We have transformed BL21(DE3) E. coli cells with a pTB146 vector to contain the gene for the CqsR CACHE domain. We grow these cells to high optical density and induce protein expression, at which point we harvest them and purify the protein. This entails lysing the cells, separating the protein with Ni-NTA resin, cleaving our protein tag, and column chromatography. With purified protein, high-throughput screens are set up to find crystallization conditions of apo CqsR, CqsR-ETA, and CqsR-AI. We then determine conditions that best lead to crystal formation and optimize them. Crystals are then diffracted with X-rays, process the data, and determine the structure of protein and AI. RESULTS/ANTICIPATED RESULTS: We anticipate finding the structure of the CqsR CACHE domain to a high resolution in addition to the identity of its autoinducer. Previous results found that the structure is homologous to another V. cholerae chemoreceptor, Mlp37, and we expect the results from this project to confirm this. In addition, we know that the autoinducer weighs approximately 62 daltons, the same as the known ligand, ethanolamine. Given that CACHE domains bind specifically to their ligands, we anticipate that the autoinducer will be structurally similar to ethanolamine. DISCUSSION/SIGNIFICANCE: The results will reveal the structure of the CqsR CACHE domain and its autoinducer. This knowledge will better allow researchers to treat cholera, as both autoinducer identity and receptor conformational changes will be uncovered, allowing for drug development to inhibit cell growth.

Type
Other
Information
Journal of Clinical and Translational Science , Volume 7 , Issue s1 , April 2023 , pp. 142 - 143
Creative Commons
Creative Common License - CCCreative Common License - BYCreative Common License - NCCreative Common License - ND
This is an Open Access article, distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is unaltered and is properly cited. The written permission of Cambridge University Press must be obtained for commercial re-use or in order to create a derivative work.
Copyright
© The Author(s), 2023. The Association for Clinical and Translational Science

Footnotes

†

Wai-Leung Ng’s name has been corrected. Additionally, middle initials for two authors have been added and the affiliations have been corrected. A corrigendum detailing these changes has been published (doi:10.1017/cts.2023.551).