Fig. 1: Identification of HPr2 and F1P as binding partners of Faecalibacterium prausnitzii FruR.

A A ligand fishing experiment was conducted to identify proteins that interact with FruR. Crude extract from F. prausnitzii A2-165 was mixed with purified 200 μg of His-FruR or His-CcpA, with protein purification buffer alone serving as a negative control. Each mixture was subjected to TALON metal affinity chromatography, and the proteins bound to His-FruR were analyzed as described in the “Methods” section. The protein size marker (Thermo Scientific, #26614) is labeled as M. The protein band corresponding to HPr2, identified by MALDI-TOF analysis, is indicated by a red arrowhead. B Purified untagged FruR (50 μg) and its mixture with each purified His-tagged form of wild-type HPr2 or the indicated phosphomimetic mutant HPr2 (50 μg) was subjected to TALON metal affinity chromatography as in panel (A). C The dissociation constants (Kd) of F1P with wild-type FruR and its mutants (5 nM each) were measured using NanoTemper Monolith NT.115pico. The Kd of F1P with the FruR(K73E) mutant and the Kd of FBP with wild-type FruR were not measurable. D The Kd of HPr2 with apo FruR, the F1P-FruR complex, and the F1P-FruR(K73E) complex were measured using NanoTemper Monolith NT.115pico. An excess of F1P (1 mM) was added to the reaction mixture to ensure full complex formation between FruR and F1P.