Datensatz

Zusammenfassung

PII signal transduction proteins have key functions in coordination of central metabolism by integrating signals from carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC7942 PII binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP- binding sites also accepting ADP. Depending on its effector molecule binding status, P II from this cyanobacterium and other oxygenic phototrophs complexes regulates the key enzyme of the cyclic ornitine pathway, N- acetyl-L-glutamate kinase (NAGK), to control arginine biosynthesis. In wild type PII E85 forms a salt bridge with R233 of NAGK, and consequently E85-PII mutants loose the ability to interact with NAGK. We found PII variants (I86N and I86T) that are able to bind to a NAGK variant (R233A) that was previously shown to be unable to bind wild type PII protein. Analysis of interactions between these P II variants and wild type NAGK as well as the NAGK R233A variant suggested that the I86N variant in the presence of ATP was a superactive NAGK binder, also indicating that PII -E85/NAGK-R233 is not essential for the interaction of the two proteins. To reveal the structural basis of this property, the crystal structure of the PII I86N variant was solved at atomic resolution. Based on the data we propose a two-step model for the mechanism of P II -NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of PII initiates the bending of the extended T-loop of PII , followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex. Crystal structures identify the binding site of 2-OG located in the vicinity between the subunit clefts and the base of the T-loop showing a novel conformation and explaining the negative effect of 2-OG on PII-NAGK interaction. Trimers with one or two 2-OG molecules shed light on the inter- subunit signalling mechanism by which P II senses effectors in a wide range of concentrations.