Fig. 5: Model of the allosteric activation pathway of the HisFH complex.

The HisFH complex adopts the inactive conformation (gray and yellow) in the apo state (left) and in the presence of one of the two substrates (Gln: red; PrFAR: blue). In the inactive conformation fL1 of HisF is in the open orientation, the HisF–HisH interface is open and the oxyanion hole is not formed. Binding of the second ligand (either PrFAR or Gln) results in the formation of the HisFH:PrFAR:Gln complex. The presence of both ligands enables the formation of the active conformation of the complex (marine and orange). This active conformation is, however, only sparsely populated and in equilibrium (indicated by red arrows) with the inactive conformation (timescale ~100 ms). In the active conformation fL1 is closed over the PrFAR substrate in the HisF active site, the HisF–HisH domains are in a closed orientation and the oxyanion hole in the HisH active site is formed, which enables Gln hydrolysis. The population of the active conformation ultimately determines the overall catalytic rate of the HisFH complex.