Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
J Biol Chem. 2021 Jan-Jun;296:100528. doi: 10.1016/j.jbc.2021.100528. Epub 2021 Mar 10.
The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.
空肠弯曲菌是一种参与宿主肠道定植和发病的细菌，其螺旋形态由肽聚糖 (PG) 层的结构决定。这种结构是由周质中 LD-羧肽酶 Pgp2 修剪肽主干决定的。Pgp2 与 PG 相互作用的界面，用于选择肽修剪的位点尚不清楚。我们确定了 Pgp2 的 1.6Å 分辨率晶体结构，其中包含一个保守的 LD-羧肽酶结构域和一个以前未表征的具有 NTF2 样折叠（NTF2）的结构域。我们在 NTF2 结构域中鉴定出一个由保守残基形成的口袋，该口袋位于 LD-羧肽酶活性位点约 40Å 处。在 pgp2 缺失菌株中转基因表达时，口袋内带电荷（Lys257、Lys307、Glu324）和疏水性残基（Phe242 和 Tyr233）的取代物不能恢复螺旋形态。寡肽分析表明，在表达这些突变体的缺失菌株中，murotripeptides 的含量减少，表明 Pgp2 催化活性降低。Pgp2 而不是 K307A 突变体被 C.jejuni Δpgp2 PG 囊泡下拉，支持口袋在 PG 结合中的作用。NMR 光谱用于定义 Pgp2 与几个 PG 片段的相互作用界面，这些片段与 LD-羧肽酶结构域内的活性位点和 NTF2 结构域的口袋结合。我们提出了一个 Pgp2 与 PG 链结合的模型，涉及 LD-羧肽酶结构域和辅助 NTF2 结构域，以诱导螺旋细胞形状。
