Extended Data Fig. 3: Determination of the necessity of selected residues for interactions between TvaE and the LP sequence of TvaA.
From: Pseudokinases can catalyse peptide cyclization through thioether crosslinking
(a) Examination of the binding affinities by ITC. TvaE or its variant was titrated with LP1–52 or its variant. (b) Conversions of 1 derivatives. Samples including 1I21R (i) and the mixtures of 1I21R with TvaE (ii), TvaF (iii) or TvaEF (iv), and 1E25R (v) and the mixtures of 1E25R with TvaE (vi), TvaF (vii) or TvaEF (viii), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1I21R, 3I21R, 1E25R, and 3E25R, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 7H]7+ (calculated 1289.1874, 1282.6212, 1286.9076, and 1280.3361, respectively. For observed details and errors, see Supplementary Figs. 25–28). The mutation sites in the precursor peptide are indicated by asterisks. (c) Conversions of 1. Samples including the mixtures of 1 with TvaF (i), TvaEF (ii), TvaEVal23GlnF (iii) and TvaEArg32GluF (iv), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1, 2 or 3, 3a and 3b (left), the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 6H]6+; and for EICs in HPLC-HR-MS analysis (right), the ESI m/z [M + 6H]6+ mode for both 2 and 3 is 1489.88. (d) Analysis of the production of thioamitide in S. coelicolor. For EIC in HPLC-HR-MS analysis, the ESI m/z [M + H]+ mode for TVA-YJ-2 is 1305.5. Tested strains include the S. coelicolor strains harboring a wild-type tva gene cluster (i) and an engineered cluster to encode mutant TvaAI21R (ii), TvaAE25R (iii), TvaEVal23Gln (iv) or TvaEArg32Glu (v), respectively. HPLC peaks corresponding to the different products are highlighted by color: green for 2, and light blue for TVA-YJ-2. Related experiments were performed >= 3 times (with >= 2 parallel samples for each).
