Molecular and structural mechanism of the gating of the porin channel

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Pseudomonas aeruginosa is a opportunistic pathogen and naturally resistant to a wide range of antibiotics. One of the mechanism for the drug resistance is that the outer membrane makes the barrier for the passage of drugs, which is due to small pores in the outer membrane. I have shown that three spices of proteins, proteins C,D2 and E1 function as a porin and all of them form the small pore. However, imipenem, a beta-lactam, which is highly effective to P.aeruginosa has been developed and clinically used. Soon after, imipenem-resistant mutants were clinically isolated and mostly lacked protein D2. In addition, protein D2 was reported to have the binding site for imipenem and basic amino acids. Then, I studied the structure and function of protein D2 and have shown that protein D2 is composed of two domains ; one is channel-forming domain and the other gate-forming domain. To search factors affecting the gating, the amino acid sequence of protein D2 was compared with other proteins and … More the region homologous to Ca^<2+>-binding proteins was demonstrated. Fluorescence of protein D2 changed by addition of Ca^<2+>, indincating the presence of Ca^<2+> binding site in protein D2. When permeability of protein D2 was measured, it was shown that the porin activity of protein D2 enhanced in the presence of Ca^<2+>. Whereas such effect of Ca^<2+> was lost by destructing the gate domain. These results suggest that Ca^<2+> ions bind to the gate domain with activation of the porin. Next, I hypothesized that protein D2 may have protease activity, which can account for the presence of the amino acid-binding site in protein D2. This possibility was investigated and the following results were obtained. (1) Purified protein D2 hydrolyzed the synthetic peptides according to Michaelis-Menten kinetics. (2) The hydrolytic reaction was inhibited by the treatment with DFP,a specific serine protease inhigitor. (3) [^3H] DFP was shown to specifically label protein D2. These results clearly indicate that the protein D2 channel has protease activity. To the best of our knowledge, this is the first reported case indicating the existence of the channel protein with portease activity. Less