New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit.
New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit.
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DOI：
10.1038/srep28664
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发表时间：
2016-06-28
影响因子：
4.6
通讯作者：
Krishnan V
中科院分区：
文献类型：
--
作者：
Chaurasia P;Pratap S;von Ossowski I;Palva A;Krishnan V
到目前为止，所有已解决的菌毛蛋白结构都来自病原菌的属和种。在这里，我们提出了来自非病原体宿主(鼠李糖乳杆菌GG)的菌毛蛋白亚基(SPAA)的第一个晶体结构。SPAA由两个串联的Cna B-型结构域组成，每个结构域都有一个异肽键和E-box基序。有趣的是，虽然N-末端结构域中的异肽键在赖氨酸和天冬酰胺之间形成，但C-末端结构域中的异肽键通常涉及天冬氨酸。我们还解决了突变蛋白质的晶体结构，其中与形成异肽键有关的残基被取代。不出所料，E-box取代的E139A突变体在N-末端区域缺乏异肽键。然而，C-端E269A替换产生了两种结构：一种是两个结构域的异肽键存在，另一种只有N-末端的结构域，但有一个未形成的异肽键和显著的构象变化。这种后一种晶体结构从未在任何其他革兰氏阳性菌毛素中观察到。值得注意的是，无论是否存在E269，D295N-取代的SPAA中仍然形成C-末端异肽键。虽然E-box突变会影响SPAA的蛋白分解和热稳定性，但没有观察到对正常菌毛聚合的累积影响。提出了SPAA在SpaCBA菌毛中聚合排列的模型。
Thus far, all solved structures of pilin-proteins comprising sortase-assembled pili are from pathogenic genera and species. Here, we present the first crystal structure of a pilin subunit (SpaA) from a non-pathogen host (Lactobacillus rhamnosus GG). SpaA consists of two tandem CnaB-type domains, each with an isopeptide bond and E-box motif. Intriguingly, while the isopeptide bond in the N-terminal domain forms between lysine and asparagine, the one in the C-terminal domain atypically involves aspartate. We also solved crystal structures of mutant proteins where residues implicated in forming isopeptide bonds were replaced. Expectedly, the E-box-substituted E139A mutant lacks an isopeptide bond in the N-terminal domain. However, the C-terminal E269A substitution gave two structures; one of both domains with their isopeptide bonds present, and another of only the N-terminal domain, but with an unformed isopeptide bond and significant conformational changes. This latter crystal structure has never been observed for any other Gram-positive pilin. Notably, the C-terminal isopeptide bond still forms in D295N-substituted SpaA, irrespective of E269 being present or absent. Although E-box mutations affect SpaA proteolytic and thermal stability, a cumulative effect perturbing normal pilus polymerization was unobserved. A model showing the polymerized arrangement of SpaA within the SpaCBA pilus is proposed.