Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA.
Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA.
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DOI：
10.1128/jb.00434-20
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发表时间：
2020-12-18
影响因子：
3.2
通讯作者：
Henderson IR
中科院分区：
文献类型：
--
作者：
Bryant JA;Cadby IT;Chong ZS;Boelter G;Sevastsyanovich YR;Morris FC;Cunningham AF;Kritikos G;Meek RW;Banzhaf M;Chng SS;Lovering AL;Henderson IR
M48金属蛋白酶广泛分布于生命的各个领域。E.大肠杆菌具有位于多个细胞区室中的该家族的四个成员。这些蛋白酶的功能还不清楚。最近的研究表明，一个家庭成员，BepA，有一个重要的作用，在成熟的一个核心组成部分的脂多糖（LPS）的生物发生机制。在这里，我们介绍了BepA的结构和结构指导的诱变策略的结果，揭示了所有M48金属蛋白酶如何发挥作用的活性所需的关键残基。不对称的革兰氏阴性外膜（OM）是细菌抵御环境侵害和抗菌剂攻击的第一道防线。OM的关键组分是脂多糖，其通过必需脂多糖转运（Lpt）系统转运至表面。Lpt系统组分LptD的正确折叠由周质金属蛋白酶BepA调节。在这里，我们提出的晶体结构的BepA从大肠杆菌，解决了2.18毫米的分辨率，其中M48蛋白酶活性位点被封闭的活性位点插头。通过我们的结构，我们表明，自由运动的活性位点插头是必不可少的BepA功能，这表明蛋白质是自动调节的活性位点插头，这是保守的整个M48金属蛋白酶家族。保守残基的靶向诱变揭示了负口袋和tetratricopeptide repeat（TPR）腔是BAM复合物组分BamA在应激条件下的功能和降解所需的。最后，我们表明，BepA的损失会导致OM脂质不对称性的破坏，导致表面暴露的磷脂。重要性M48金属蛋白酶广泛分布于生命的各个领域。E.大肠杆菌具有位于多个细胞区室中的该家族的四个成员。这些蛋白酶的功能还不清楚。最近的研究表明，一个家庭成员，BepA，有一个重要的作用，在成熟的一个核心组成部分的脂多糖（LPS）的生物发生机制。在这里，我们介绍了BepA的结构和结构指导的诱变策略的结果，揭示了所有M48金属蛋白酶如何发挥作用的活性所需的关键残基。
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. 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 Å, 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.