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首页> 外文期刊>Journal of bacteriology >Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA
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Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA

机译：大肠杆菌M48金属蛋白酶BEPA保守调节机制的结构功能特征

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The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18??, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.
机译：不对称革兰负外膜（OM）是对环境侮辱和抗菌药物攻击的细菌的第一道防线。 OM的关键组分是脂多糖，通过必需的脂多糖输送（LPT）系统将其运输到表面。 LPT系统组分LPTD的正确折叠由周质金属蛋白酶，BEPA调节。这里，我们介绍了来自大肠杆菌的BEPA的晶体结构，解决了2.18的分辨率，其中M48蛋白酶活性位点被活性位点插头堵塞。我们的结构知情，我们证明了活性位点插头的自由流动对于BEPA功能至关重要，表明蛋白质通过活性位点塞自动造成，这在整个M48金属蛋白酶家族中保守。保守残余物的靶向诱变表明，在应激条件下，BAM复合成分BAMA的功能和降解所需的负口袋和四氢肽重复（TPR）腔。最后，我们表明BEPA的丧失导致OM脂质不对称的破坏，导致表面暴露的磷脂。 Importance M48金属蛋白酶在生命的所有领域中广泛分布。大肠杆菌拥有这个家庭的四名成员，位于多个蜂窝隔间。这些蛋白酶的功能尚不清楚。最近的调查显示，一个家庭成员Bepa在脂多糖（LPS）生物生成机械的中央组分的成熟中具有重要作用。在这里，我们提出了BEPA的结构和结构引导诱变策略的结果，其揭示了活动所需的关键残留物，可通知所有M48金属蛋白释放酶的功能。

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