Fig. 3: Structure guided mutagenesis and homolog screening reveals hinT mutants with increased affinity. | Nature Communications

Fig. 3: Structure guided mutagenesis and homolog screening reveals hinT mutants with increased affinity.

From: A repurposed AMP binding domain reveals mitochondrial protein AMPylation as a regulator of cellular metabolism

Fig. 3

A Multiple sequence alignment of E. coli, T. marianensis, and H. sapiens hinT. Conserved amino acids are highlighted in light purple (two of three homologs) and dark purple (in all three homologs).The conserved histidine triad motif is noted with the histidine corresponding to E. coli His101 labeled in red. Amino acids present in the variable C termini of homologs are boxed in orange. B–DRepresentative BLI sensorgrams depicting the binding response of serial dilutions of 500 nM to 7.8 nM AMPylated sucA to immobilized GST-E. coli hinT (B), GST-T. marianensis hinT (C), and GST-H. sapiens hinT (D). E Steady state binding response of sucA-AMP to immobilized GST-hinT homologs from BLI experiment. F Binding affinities measured from the steady state binding response of GST-hinT homologs to sucA-AMP. The KD values were determined from steady state binding responses as a non-linear regression curve and reported with a 95% confidence interval. Source data are provided as a Source Data file.

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