Molecular and structural mechanism of the gating of the porin channel

铜绿假单胞菌是一种条件致病菌，对多种抗生素具有天然耐药性。耐药的机制之一是外膜形成药物通过的屏障，这是由于外膜中的小孔。我已经证明了三种蛋白质，蛋白质C，D2和E1作为孔蛋白发挥作用，它们都形成了小孔。然而，对铜绿假单胞菌高度有效的β-内酰胺亚胺培南已被开发并临床使用。不久之后，临床上分离出亚胺培南耐药突变体，大多缺乏蛋白D2。此外，蛋白D2被报道具有亚胺培南和碱性氨基酸的结合位点。然后，研究了蛋白D2的结构和功能，发现蛋白D2由两个结构域组成，一个是通道形成结构域，另一个是门形成结构域。为了寻找影响门控的因素，将蛋白D2的氨基酸序列与其他蛋白进行比较， ...更多信息 证明了与Ca^2+结合蛋白同源的区域。加入Ca^<2+>后，蛋白D2的荧光发生了变化，表明蛋白D2中存在Ca^<2+>结合位点。当测量蛋白D2的渗透性时，显示蛋白D2的孔蛋白活性在Ca^<2+>存在下增强。而Ca^<2+>的这种作用则因栅畴的破坏而丧失.这些结果表明，Ca^2+离子与门结构域结合，激活了孔蛋白。接下来，我假设蛋白质D2可能具有蛋白酶活性，这可以解释蛋白质D2中氨基酸结合位点的存在。对这种可能性进行了研究，并获得了以下结果。(1)纯化的蛋白D2根据Michaelis-Menten动力学水解合成肽。(2)用丝氨酸蛋白酶抑制剂DFP处理可抑制水解反应。(3)[^3H] DFP显示特异性标记蛋白D2。这些结果清楚地表明蛋白D2通道具有蛋白酶活性。据我们所知，这是第一个报告的情况下，表明存在的通道蛋白与portease活性。少

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