1H, 13C and 15N assignment of self-complemented MrkA protein antigen from Klebsiella pneumoniae

IF 0.8 4区 生物学 Q4 BIOPHYSICS Biomolecular NMR Assignments Pub Date : 2024-07-17 DOI:10.1007/s12104-024-10185-3
Valentina Monaci, Gianmarco Gasperini, Lucia Banci, Francesca Micoli, Francesca Cantini
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Abstract

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein’s N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the 13C,15N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.

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肺炎克雷伯氏菌自补体 MrkA 蛋白抗原的 1H、13C 和 15N 赋值。
肺炎克雷伯氏菌(Kp)对公共卫生的威胁日益严重,特别是考虑到它与医院内感染的关系,以及它已成为新生儿败血症的主要病因,尤其是在中低收入国家(LMICs)。Kp 的宿主细胞粘附和生物膜形成是由 1 型和 3 型缘膜介导的,其主要缘膜亚基分别由 fimA 和 mrkA 基因编码。在本研究中,我们重点研究了MrkA亚基,它是一种20 KDa的蛋白质,其三维分子结构仍然难以捉摸。我们利用溶液核磁共振分析了一种重组版本的 MrkA,其中位于蛋白质 N 端的供体链段被移至 C 端,之前是一个六甘氨酸连接体。这种构建物产生了一种自补体的 MrkA 变体。值得注意的是,自补的 MrkA 单体失去了与其他单体相互作用的能力,也无法延伸到流苏结构中。在这里,我们报告了 13C、15N 标记的自补体 MrkA 单体的近乎完整的分配。此外,对其内部迁移率的研究发现,除了环 176-181 中富含甘氨酸的区域外,整个多肽序列的弛豫参数基本一致。这些数据为全面阐明MrkA单体的结构以及绘制MrkA与抗原诱导抗体之间分子相互作用区域的结构图铺平了道路。
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来源期刊
Biomolecular NMR Assignments
Biomolecular NMR Assignments 生物-光谱学
CiteScore
1.70
自引率
11.10%
发文量
59
审稿时长
6-12 weeks
期刊介绍: Biomolecular NMR Assignments provides a forum for publishing sequence-specific resonance assignments for proteins and nucleic acids as Assignment Notes. Chemical shifts for NMR-active nuclei in macromolecules contain detailed information on molecular conformation and properties. Publication of resonance assignments in Biomolecular NMR Assignments ensures that these data are deposited into a public database at BioMagResBank (BMRB; http://www.bmrb.wisc.edu/), where they are available to other researchers. Coverage includes proteins and nucleic acids; Assignment Notes are processed for rapid online publication and are published in biannual online editions in June and December.
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