Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli
Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli
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DOI：
10.1074/jbc.274.10.6726
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发表时间：
1999-03-05
影响因子：
4.8
通讯作者：
Höltje, JV
中科院分区：
文献类型：
--
作者：
Vollmer, W;von Rechenberg, M;Höltje, JV
细菌胞壁蛋白囊的形成依赖于胞壁蛋白水解酶和胞壁蛋白水解酶的协同作用。为了了解这两类蛋白质的相互作用机制，进行了亲和层析和表面等离子体共振（SPR）研究。膜结合的裂解转糖基酶MltA共价连接到溴化氰活化的琼脂糖凝胶特异性保留青霉素结合蛋白（PBPs）1B，1C，2和3从大肠杆菌的粗Triton X-100膜提取物。在周质蛋白的存在下，PBP 1A也被特异性结合。至少5种不同的非PBPs对MltA-Sepharose具有特异性。在E.大肠杆菌基因组。这种MltA相互作用蛋白，命名为MipA，另外结合PBP 1B，一种双功能胞壁蛋白转糖基酶/转肽酶。将PBP 1B固定在氨苄青霉素包被的传感器芯片上的SPR研究显示PBP 1B的寡聚化，这可能表明二聚化。将MipA和MltA同时施加到PBP 1B传感器芯片表面上导致三聚复合物的形成，解离常数被确定为约10（-6）M，在MipA存在下，胞壁素聚合酶（PBP 1B）和胞壁素水解酶（MltA）之间形成复合物代表了重构假设的合成胞壁素的全酶的第一步，推测可能是E.杆菌
Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/ transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization, Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex, The dissociation constant was determined to be about 10(-6) M, The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.