SASDBH4: Wild-type LytA (N-His6) without choline (Lytic Amidase without ch...

基本情報

• 登録情報: データベース: SASBDB / ID: SASDBH4

試料

Wild-type LytA (N-His6) without choline

• Lytic Amidase without choline (protein), wt-LytA-His6, Streptococcus pneumoniae

• 生物種: Streptococcus pneumoniae (肺炎レンサ球菌)
• 引用: ジャーナル: mBio / 年: 2014; タイトル: Structural and functional insights into peptidoglycan access for the lytic amidase LytA of Streptococcus pneumoniae.; 要旨: The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs.; IMPORTANCE: Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

登録者

• Al Kikhney (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

モデル

• モデル #503: ; タイプ: atomic / ソフトウェア: SASREF (7) / カイ2乗値: 2.053489; Omokage検索でこの集合体の類似形状データを探す (詳細)

試料

• 試料: 名称: Wild-type LytA (N-His6) without choline / 試料濃度: 1.00-4.00
• バッファ: 名称: Tris / 濃度: 20.00 mM / pH: 7.5 / コメント: no choline / 組成: 150 mM NaCl, 1 µM ZnCl2
• 要素 #327: 名称: wt-LytA-His6 / タイプ: protein / 記述: Lytic Amidase without choline / 分子量: 37.4 / 分子数: 1 / 由来: Streptococcus pneumoniae

実験情報

• ビーム: 設備名称: DORIS III X33 / 地域: Hamburg / 国: Germany / 形状: 0.6 / 線源: X-ray synchrotron / 波長: 0.15 Å / スペクトロメータ・検出器間距離: 2.7 mm
• 検出器: 名称: Pilatus 1M-W / Pixsize x: 0.172 mm

スキャン

タイトル: wt-LytA-His6 no choline / 測定日: 2011年7月3日 / セル温度: 10 °C / 照射時間: 15 sec. / フレーム数: 8 / 単位: 1/nm /

	Min	Max
Q	0.1765	6.0241

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 773 /

	Min	Max
Q	0.250352	2.36457
P(R) point	1	773
R	0	12.5

結果

カーブのタイプ: extrapolated / Standard: BSA
コメント: Without choline LytA is monomeric in solution.

	実験値	Experimental error	Standard	Porod	Porod error
分子量	40 kDa	4	66 kDa	35 kDa	4
体積	-	-	-	56 nm3	-

	Guinier	P(R)
前方散乱 I0	39.83	-
慣性半径, Rg	3.36 nm	3.655 nm

	Min	Max	Error
D	-	12.5	1.3
Guinier point	28	75	-