Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions

In this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1–98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content.PcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia.Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium which infects immunocompromised individuals, such as those suffering from AIDS, cancer, and severe burns; in addition, it is highly prevalent in the lungs of patients with cystic fibrosis (CF), being responsible for a large number of deaths in such cases [1]. P. aeruginosa, like most other Gram-negative pathogens, employs a type III secretion system (TTSS) to inject toxins into the cytoplasm of contacted host cells [2]. Such a system requires more than 20 different proteins, a number of which are assembled into a "secreton" structure wh