Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
J Biol Chem. 2023 Mar;299(3):103001. doi: 10.1016/j.jbc.2023.103001. Epub 2023 Feb 9.
The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with K values in the low millimolar range. Since these K values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.
VanRS 双组份系统调节万古霉素耐药肠球菌的耐药表型。VanS 是一种感应组氨酸激酶，通过自身磷酸化并随后将磷酸基团转移到响应调节子 VanR 来响应万古霉素的存在。磷酸转移激活 VanR 作为转录因子，从而启动耐药基因的表达。VanS 蛋白的结构信息仍然难以捉摸，阻碍了对其功能的分子水平理解。在这里，我们展示了源自万古霉素耐药肠球菌 A 型和 C 型的两种 VanS 蛋白的催化和 ATP 结合 (CA) 结构域的 X 射线晶体结构。这两种蛋白质都采用了经典的 Bergerat 折叠，这种折叠在其他原核组氨酸激酶的 CA 结构域中也观察到过。我们试图确定这两种蛋白质的核苷酸结合形式的结构；然而，尽管反复努力，这些形式仍无法结晶，促使我们测量蛋白质与 ATP 的结合亲和力。出乎意料的是，两个 CA 结构域对核苷酸的亲和力都很低，K 值在低毫摩尔范围内。由于这些 K 值与细胞内 ATP 浓度相当，这种弱的底物结合可能反映了一种调节耐药表型表达的方式。
