Isolation and Characterization of the Phage T4 PinA Protein, an Inhibitor of the ATP-dependent Lon Protease ofEscherichia coli

Abstract

The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit M r of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (K d ϳ 10 nM for dimer binding), and a complex with ϳ1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65°C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease.

Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates

“…For example, unfolded casein stimulates both the ATPase and peptidase activity of Lon by interacting with an allosteric protein-binding site on Lon (82)(83)(84). In addition, the bacteriophage T4 PinA protein specifically inhibits E. coli Lon but not ClpAP or HslUV (ClpYQ) (82). PinA does not interact with the ATP-binding site, the proteolytic active site or the allosteric protein-binding site of Lon.…”

“…Unfolded protein substrates have been shown to stimulate both the peptidase and ATPase activities of Lon (2,81). For example, unfolded casein stimulates both the ATPase and peptidase activity of Lon by interacting with an allosteric protein-binding site on Lon (82)(83)(84). In addition, the bacteriophage T4 PinA protein specifically inhibits E. coli Lon but not ClpAP or HslUV (ClpYQ) (82).…”

PinA Inhibits ATP Hydrolysis and Energy-dependent Protein Degradation by Lon Protease

“…PinA and Lon, assay for casein degradation, buffer composition, and methods for SDS-PAGE 1 and gel filtration were described in the preceding paper (13). An ATP-regenerating system consisting of 50 mM creatine phosphate and 20 g/ml phosphocreatine kinase was used in some assays.…”

“…Our previous data showed that a dimer of PinA binds to a tetramer of Lon (13). Lon has four potential ATPase sites per tetramer, and inhibition of all stimulated ATPase activity by a single PinA dimer implies that Lon subunits act in a cooperative manner.…”

“…In the preceding paper, we showed that purified PinA forms a complex with Lon protease and inhibits its casein degradation activity in vitro (13). Here, we investigate the mechanism of Lon inhibition by PinA and suggest that PinA interferes with the coupling between ATP hydrolysis and protein degradation, possibly by preventing protein unfolding by Lon.…”