Characterization of a Salmonella Transcription Factor-DNA Complex and Identification of the Inducer by Native Mass Spectrometry

Blake E Szkoda; Angela Di Capua; Joy Shaffer; Edward J Behrman; Vicki H Wysocki; Venkat Gopalan

doi:10.1016/j.jmb.2022.167480

Characterization of a Salmonella Transcription Factor-DNA Complex and Identification of the Inducer by Native Mass Spectrometry

J Mol Biol. 2022 Apr 15;434(7):167480. doi: 10.1016/j.jmb.2022.167480. Epub 2022 Feb 14.

Authors

Blake E Szkoda¹, Angela Di Capua², Joy Shaffer³, Edward J Behrman³, Vicki H Wysocki⁴, Venkat Gopalan⁵

Affiliations

¹ Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
² Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University, Columbus, OH 43210, USA.
³ Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
⁴ Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA; Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA. Electronic address: wysocki.11@osu.edu.
⁵ Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA. Electronic address: gopalan.5@osu.edu.

Abstract

FraR, a transcriptional repressor, was postulated to regulate the metabolism of the Amadori compound fructose-asparagine (F-Asn) in the foodborne pathogen Salmonella enterica. Here, the DNA- and inducer-binding affinities and stoichiometries of FraR were determined and cross-validated by electrophoretic mobility-shift assays (EMSAs) and online buffer exchange coupled to native mass spectrometry (OBE-nMS). We demonstrate the utility of OBE-nMS to characterize protein and protein-DNA complexes that are not amenable to offline exchange into volatile buffers. OBE-nMS complemented EMSAs by revealing that FraR binds to the operator DNA as a dimer and by establishing 6-phosphofructose-aspartate as the inducer that weakens DNA binding by FraR. These results provide insights into how FraR regulates the expression of F-Asn-catabolizing enzymes and add to our understanding of the intricate bacterial circuitry that dictates utilization of diverse nutrients.

Keywords: Samonella FraR; fructose-asparagine utilization; native mass spectrometry; transcriptional regulation.

MeSH terms

Asparagine / metabolism
Bacterial Proteins* / metabolism
DNA / metabolism
Fructose / metabolism
Mass Spectrometry / methods
Salmonella enterica* / metabolism
Transcription Factors* / metabolism

Substances

Bacterial Proteins
Transcription Factors
Fructose
Asparagine
DNA

Abstract

MeSH terms

Substances

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