Fig. 7: The conformational dynamics in AcuB depends on the nucleotide-loading state and affects interaction of AcuC.

a GsAcuB inhibits GsAcuC in presence of AMP but not ADP, ATP, or Ap4A. IC50 values were determined by an FdL assay using Boc-Lys(Ac)-AMC as a substrate. The relative fluorescence of the released coumarin fluorophore was determined and is shown in arbitrary units (a.u.). The experiment was performed in three technical replicates (n = 3). Data are presented as mean ± SD. Source data are provided as Source Data file. b AlphaFold3 model of a BsAcuB₂•2BsAcuC heterotetramer with four molecules of AMP bound to the AcuB dimer. Two molecules of BsAcuC bind to the BsAcuB dimer. Each AcuC molecule forms major contacts to the ACT domain of one monomer and additionally contacts the Bateman domain of the other monomer. This creates an interaction site consisting of two contact sites of AcuC on both monomers of the AcuB dimer, i.e., conformational changes in the scissor-shaped AcuB dimer either altering the distance of an ACT domain to a Bateman domain or their relative orientation might directly affect the binding affinity of AcuC towards AcuB. The figure was created with PyMOL⁸⁸. c GsAcuB M189R is unable to inhibit GsAcuC and GsAcuB H87W has a reduced inhibitory potency compared to GsAcuB wild type. IC₅₀ values were determined by a FdL assay using Boc-Lys(Ac)-AMC as a substrate. The relative fluorescence of the released coumarin fluorophore was determined and is shown in arbitrary units (a.u.). The experiment was performed in three technical replicates (n = 3). The curves shown for GsAcuB wild type with AMP/without nucleotide are the same as shown in a. Shown are the mean-values and standard deviations. Source data are provided as Source Data file.