The complex assembly of N-acetyl-L-glutamate kinase with the PII signal transduction protein follows a two-step mechanism

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. Through biochemical and biophysical analyses, we show that the PII variant lacking the T-loop (PII _ΔT-loop ) is blocked in the first association step with NAGK, but is still able to form a stable complex. However, the NAGK interaction with PII _ΔT-loop is weaker than with the wild-type PII as indicated by a decrease in the affinity. Using single particle cryo-EM, we resolved the structure of the encounter PII _ΔT-loop -NAGK complex, revealing that the PII _ΔT-loop -NAGK interface 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.