Filamentation and proline inhibition of glutamate kinase

Glutamate kinase (GK) is the first committed enzyme in the proline biosynthesis pathway. Belonging to amino acid kinase (AAK) superfamily, most prokaryotic GKs have an additional PseudoUridine synthase and Archaeosine transglycosylase (PUA) domain at the C-terminus, while the function of the PUA domain in GK is poorly understood. Here, we find that Escherichia coli GK (EcGK) assembles into filaments and bundles in the state of apo and proline binding. Using cryogenic electron microscopy, we determine the high-resolution structures of EcGK filaments and bundles. The PUA domain is necessary for EcGK filaments and bundles, and the main interfaces have been clearly defined. The feedback inhibitor proline binds at the same pocket as substrate glutamate, inducing conformational changes on nearby regulatory loop which facilitate proline binding. The PUA domain stabilizes the regulatory loop and contributes to proline feedback inhibition. This study reports the special filament-based assembly of EcGK at apo and proline binding state. The first proline binding structure in the GK family illustrates the feedback inhibition mechanism. Intriguingly, the PUA domain is involved in both filamentation and feedback inhibition of EcGK, revealing the versatility of this ancient domain.