## 3 Materials and Methods
### Experimental Methods  
**Cloning, Expression, and Purification of Rel\({}_{Seq}\) constructs. ** A pET21 expression vector containing the DNA sequence coding for Rel\({}_{Seq}\) 1-385 fused with a C-terminal His-tag was purchased from Giotto Biotech. Two truncated constructs of the bifunctional enzyme, Rel\({}_{Seq}\) 79-385 (Rel\({}_{Seq}\) SYNTH) and Rel\({}_{Seq}\) 1-224 (Rel\({}_{Seq}\) HYD) [50], presenting only the synthetase or hydrolase domain, respectively, were obtained with the Q5 Site-directed mutagenesis kit (New England Biolabs). Each protein construct was overproduced in BL21(DE3) _Escherichia coli_ cells (Merck), grown in LB medium. Protein expression was induced by the addition of 0.5 mM IPTG and prolonged overnight at 25 \({}^{\circ}\)C for Rel\({}_{Seq}\) 1-385 and Rel\({}_{Seq}\) HYD and at 20 \({}^{\circ}\)C for Rel\({}_{Seq}\) SYNTH. In a typical purification, bacterial cells harvested by centrifugation were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 10 mM imidazole, 0.5 mM TCEP), supplemented with 1 mM phenylmethanesulfonylfluoride, 20 \(\upmu\)g/mL DNAse I (Merck) and, only in the case of Rel\({}_{Seq}\) SYNTH, 0.1% Triton X-100. Cell disruption was performed by sonication, and, after high-speed centrifugation and microfiltration, the resulting bacterial soluble extract was loaded on two 1 mL Ni Sepharose HisTrap columns (GE Healthcare), connected in series and equilibrated with lysis buffer. Elution of Rel\({}_{Seq}\) constructs was achieved by applying a linear gradient of elution buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 500 mM imidazole, 0.5 mM TCEP) over 15 column volumes. After a size exclusion chromatography step on a HiPrep 16/60 Sephacryl S-200 HR (GE Healthcare), Rel\({}_{Seq}\) constructs were stored at \(-80\) \({}^{\circ}\)C in 50 mM Tris-HCl pH 8, 200 mM NaCl, 5% glycerol. **Thermal shift assays on selected fragments vs. Rel\({}_{Seq}\) constructs. ** The binding of the selected fragments to Rel\({}_{Seq}\) constructs was assessed by titration of the protein in thermal shift assays, performed using a Step One Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Assays were performed in 20 mM Tris-HCl, pH 8, containing 150 mM NaCl. The final protein concentration was kept at 0.5 mg/mL for all Rel\({}_{Seq}\) constructs, and the fluorescent Protein Thermal Shift Dye (Thermo Fisher Scientific, Waltham, MA, USA) was used to monitor protein unfolding within the excitation/emission ranges 470-505/540-700 nm. Each fragment was dissolved in DMSO at a stock concentration of 200 mM, and two-fold dilution series were prepared to have final compound concentrations ranging from 0.3 mM to 10 mM; 2.5% DMSO was added in place of the fragments for control samples. Assays were performed in triplicate at a final volume of 15 \(\upmu\)L in MicroAmp\({}^{\text{TM}}\) Fast Optical 48-well reaction plates (Thermo Fisher Scientific, Waltham, MA, USA) sealed with adhesive films. Plates were heated from 25 to 90 \({}^{\circ}\)C with a heating rate of 0.5 \({}^{\circ}\)C/min. The \(K_{\text{d}}\) of protein-ligand complexes engaged by Rel\({}_{Seq}\) constructs with the tested fragments was calculated from the plot of protein melting temperature variations as a function of fragmentation concentrations with the equation for Ligand Binding (1 site) provided with the software GraFit 5.0 (Erihtacus Software, Staines, UK). **STD-NMR experiments on selected fragments vs. Rel\({}_{Seq}\) constructs. ** Experiments were performed on a 600 MHz Bruker Avance spectrometer. All experiments were acquired at 298 and 283 K on the free ligands in deuterated phosphate buffer pH 7.4. A DMSO-d6 percentage of about 5% was added to dissolve the fragments. In 1D spectra, water suppression was achieved by excitation sculpting sequence. STD NMR experiments were performed using WATERGATE 3-9-19 pulse sequence for water suppression.