Structural basis of cell wall cleavage by a staphylococcal autolysin.
Structural basis of cell wall cleavage by a staphylococcal autolysin.
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DOI：
10.1371/journal.ppat.1000807
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发表时间：
2010-03-12
期刊：
影响因子：
6.7
通讯作者：
Stehle T
中科院分区：
文献类型：
--
作者：
Zoll S;Pätzold B;Schlag M;Götz F;Kalbacher H;Stehle T
表皮葡萄球菌和金黄色葡萄球菌的主要自溶素（Atl）在细胞分离中发挥重要作用，其突变体的毒力也减弱。因此，自溶素代表了新型抗生素开发的有希望的目标。在这里，我们报告了来自表皮葡萄球菌主要自溶素的催化活性酰胺酶结构域 AmiE（表皮葡萄球菌酰胺酶）的高分辨率结构。这是第一个具有酰胺酶样折叠的蛋白质结构，来自具有革兰氏阳性细胞壁结构的细菌。 AmiE 采用球状折叠，多个 α 螺旋围绕中央 β 折叠。序列比较揭示了一组保守氨基酸，这些氨基酸定义了与埋藏锌离子的假定结合位点。假定活性位点中关键残基的突变会导致活性丧失，使我们能够提出一种催化机制。我们还鉴定并合成了胞壁酰三肽，它是可用作酶底物的最小肽聚糖片段。胞壁酰三肽衍生物的分子对接和消化测定使我们能够识别配体结合的关键决定因素。这产生了该配体相互作用的合理模型，不仅适用于 AmiE，而且适用于具有相同折叠的其他 PGN 水解酶。由于 AmiE 活性位点突变也显示出严重的生长缺陷，因此我们的研究结果为设计针对葡萄球菌细胞分离的特异性抑制剂提供了极好的平台，从而可以防止这种病原体的生长。尽管葡萄球菌是人类皮肤和呼吸道的常见寄生菌，但许多高致病性菌株是医院相关感染的主要原因，并且可能危及生命，尤其是免疫功能低下的患者。此外，越来越多的菌株对常用抗生素产生了耐药性，这给感染的治疗带来了挑战。因此，需要作用于新靶点的抗菌药物来对抗多重耐药葡萄球菌的进一步传播。自溶素是一种细胞壁相关酶，对细胞增殖至关重要，代表了一个有希望的新靶点。我们使用 X 射线晶体学以原子分辨率解析了表皮葡萄球菌自溶素 AmiE 催化活性区域的结构。我们的研究揭示了蛋白质表面上特定细胞壁成分的明确结合凹槽。使用计算机计算与生化研究相结合，我们能够识别自溶素识别细胞壁所需的关键基序。我们的数据进一步表明，除了这些核心基序之外，细菌细胞壁的物种特异性改变是配体的明确识别的原因。了解酶-底物复合物中的相互作用以及有关催化机制的信息是成功开发抗菌药物的先决条件。因此，我们的结果提供了一个可以推出新型抑制剂的平台。
The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several α-helices surrounding a central β-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen. Although Staphylococci are common habitants of the human skin and the respiratory tract, a number of highly pathogenic strains are a major cause of hospital-associated infections and can be life threatening especially in immunocompromised patients. Moreover, an increasing number of strains has acquired resistance against commonly used antibiotics, which makes treatment of infections a challenge. Therefore, antibacterial drugs that act on new targets are needed to counteract the further spread of multiresistent staphylococci. The autolysins, which are cell wall associated enzymes that are essential for cell proliferation, represent one promising such new target. We used x-ray crystallography to solve the structure of a catalytically active region of the autolysin of Staphylococcus epidermidis, AmiE, at atomic resolution. Our studies reveal a defined binding groove for a specific cell wall component on the protein surface. Using in silico calculations in combination with biochemical studies, we are able to identify key motifs that are required for the recognition of the cell wall by autolysins. Our data further indicate that, besides these core motifs, species-specific alterations of bacterial cell walls are responsible for the unambiguous identification of ligands. Knowledge of the interactions in an enzyme-substrate complex, as well as information about the mechanism of catalysis, are prerequisites for the successful development of antimicrobial drugs. Our results therefore provide a platform from which a new class of inhibitors can be launched.
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影响因子：
5.6
作者：
Guan, RJ;Wang, Q;Mariuzza, RA
通讯作者：
Mariuzza, RA
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.6
作者：
Heilmann, C;Schweitzer, O;Gotz, F
通讯作者：
Gotz, F
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
2.1
作者：
Biswas, Raja;Voggu, Lalitha;Goetz, Friedrich
通讯作者：
Goetz, Friedrich