Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria
Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria
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DOI：
10.1002/pro.5002
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发表时间：
2024-06-01
期刊：
影响因子：
8
通讯作者：
Ikegami，Takahisa
中科院分区：
文献类型：
--
作者：
Konuma，Tsuyoshi;Takai，Tomoyo;Ikegami，Takahisa
对大多数抗生素产生抗药性的细菌，特别是那些引起医院感染的细菌，会造成严重的问题。其中，万古霉素耐药肠球菌的出现是一个巨大的冲击，因为万古霉素是控制耐甲氧西林金黄色葡萄球菌的最后手段。因此，迫切需要开发一种与万古霉素耐药有关的蛋白VanX的抑制剂。虽然VanX的晶体结构已经被解析，但它的不对称单元包含六个连续排列的分子。我们建立了VanX在溶液中作为稳定二聚体的结构模型，主要是利用核磁共振(核磁共振)残留偶极耦合。尽管二聚体的分子质量为46 kDa，但分析通常不像对约10 kDa的小蛋白那样直接，但成功地进行了分析。我们用氨基酸选择性非标记法对主链进行了归属。因为我们发现二聚体结构中的锌离子配位活性中心位于与二聚体界面相反的方向，所以我们通过替换二聚体界面上的一个氨基酸来生成活性单体。该单体仅由202个氨基酸组成，有望在未来的研究中使用，以利用核磁共振筛选和改进抑制剂。
Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin‐resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin‐resistantStaphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid‐selective unlabeling method. Because we found that the zinc ion‐coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.