Abstract
<jats:p>PII signal transduction proteins control central carbon/nitrogen metabolism via interacting with and modulating many cellular targets. In photoautotrophs, N-acetyl-L-glutamate kinase (NAGK), the rate-limiting enzyme of arginine biosynthesis, is the primary target of PII. Here, we focused on how PII initiates the interaction with NAGK to unravel the molecular mechanism of PII-NAGK complex assembly and PII-induced NAGK activation. Through biochemical and biophysical analyses, we show that the PII variant lacking the T-loop (PIIΔT-loop) is blocked in the first association step with NAGK, but is still able to form a stable complex. However, the NAGK interaction with PIIΔT-loop is approximately two times weaker than with the wild-type PII as indicated by an increase in the Kd value (≈ 50%) in the presence or absence of PII/NAGK effector molecules. The enzymatic activity of NAGK in complex with PIIΔT-loop is also reduced compared to the wild-type PII protein. Using single particle cryo-EM, we elucidated the structure of the encounter PIIΔT-loop-NAGK complex, revealing that the PIIΔT-loop-NAGK interface in the ligand-bound complex is established by the B-loop residues of PII. Thus, our data indicate a two-step process of PII-NAGK complex formation with B-loop initiating the contact with NAGK followed by the insertion of the PII T-loop deeply into the NAGK cavity to fully activate the enzyme. By defining the molecular mechanism of the encounter complex and its impact on NAGK architecture, ligand binding, and activation, our study provides essential details on how the PII-NAGK complex is assembled and regulated in response to the cell's metabolic state.</jats:p>