Characterization of Transcription Factor‐DNA Complexes Using Online Buffer Exchange Coupled to Native Mass Spectrometry

Abstract

The foodborne pathogen Salmonella enterica serovar Typhimurim (Salmonella) causes approximately 94 million enteric infections and 50,000 diarrheal deaths annually worldwide. The Center for Disease Control and Prevention estimates 1.35 million Salmonella-related illnesses in the United States annually. There are no vaccines or antibiotics to specifically combat this bacterium. During inflammation post-infection, Salmonella exploits fructose-asparagine (F-Asn) as a carbon and nitrogen source. F-Asn–formed during cooking or dehydration of raw foods–is a product of an Amadori rearrangement following the non-enzymatic condensation of glucose and asparagine. F-Asn is metabolized using three enzymes and a transporter encoded by the fra operon. The roles of a periplasmic asparaginase (FraE), cytoplasmic kinase (FraD), and a cytoplasmic deglycase (FraB) in F-Asn catabolism are now established. Importantly, the FraB deglycase was identified as a promising drug target because FraB dysfunction led to accumulation of its substrate and self-poisoning of Salmonella. The current work was undertaken to characterize gene regulation of the fra operon by FraR, the putative transcription factor in this locus. FraR is predicted to be a member of the GntR transcription factor family with an N-terminal DNA-binding domain (DBD) and a C-terminal inducer-binding domain (IBD). We hypothesize that FraR binds to the fraB promoter in vivo and acts as a transcriptional repressor, and that binding of an inducer to the FraR-IBD triggers a conformational change to release the DNA from the DBD and thereby permit transcription of the fra genes. This postulate was tested by first purifying recombinant FraR (post-overexpression in Escherichia coli), and then assessing FraR-DNA binding affinity (± putative inducers) using two cross-validating methods: fluorescence-based gel-shift assays and online buffer exchange (OBE) coupled to native mass spectrometry (nMS). With OBE, samples are kept in a non-volatile buffer that favors their native biological properties and then buffer exchanged into ammonium acetate on-line for MS analysis. Thus, OBE-nMS eliminates difficulties typically associated with sample preparation and expedites characterization of protein and protein-DNA complexes. Here, we used OBE-nMS to confirm the oligomeric state of FraR and FraR-DNA complexes and to investigate 6-phospho-fructose-aspartate (6-P-F-Asp) as a potential inducer. Results from our studies have provided insights into the binding stoichiometry of FraR, operator sequence, and inducer identity. We showed that a FraR dimer binds with high affinity to two 26-bp DNA fragments (KD ~1 nM), and that two 6-P-F-Asp molecules bind to each dimer (KD ~2 µM). Our studies have also shown that 6-P-F-Asp acts as the inducer that triggers FraR dissociation from the DNA. These findings provide a first glimpse into the regulation of Amadori metabolism in a clinically significant bacterial pathogen.