The complex assembly of N-acetyl-L-glutamate kinase with the PII signal transduction protein follows a two-step mechanism
Year: 2026
Type: preprint
Abstract: ABSTRACT 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.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: Deutsche Forschungsgemeinschaft
Awards: SFB 1535: Mikrobielle Netzwerke – von Organellen bis hin zu Reichübergreifenden Lebensgemeinschaften, G1514386576, G1800357742, G1968701140, G2036822895, G2691370021, EXC 2124: Kontrolle von Mikroorganismen zur Bekämpfung von Infektionen (CMFI), G6625650684, Multiproteinkomplexe, die die ChlorophyllBiosynthese steuern, SFB 1381: Dynamische Organisation zellulärer Proteinmaschinerien: Von der Biogenese und modularen Assemblierung zur Funktion
OpenAlex ID: W7202124450