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-基本情報
登録情報 | データベース: PDB / ID: 6z05 | ||||||
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タイトル | Campylobacter jejuni serine protease HtrA | ||||||
要素 | DegQ family serine endoprotease | ||||||
キーワード | LYASE / Campylobacter jejuni / serine protease / HtrA / dodecamer | ||||||
機能・相同性 | 機能・相同性情報 | ||||||
生物種 | Campylobacter jejuni (カンピロバクター) | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 5.8 Å | ||||||
データ登録者 | Grinzato, A. / Kandiah, E. / Zanotti, G. | ||||||
引用 | ジャーナル: Gut Microbes / 年: 2020 タイトル: Functional analysis and cryo-electron microscopy of serine protease HtrA. 著者: Urszula Zarzecka / Alessandro Grinzato / Eaazhisai Kandiah / Dominik Cysewski / Paola Berto / Joanna Skorko-Glonek / Giuseppe Zanotti / Steffen Backert / 要旨: is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA ), which targets tight ... is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA by comparison to the HtrA counterparts from and . We defined the architecture of HtrA at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA . Together, this is the first report on the in-depth functional and structural analysis of HtrA , which may allow the construction of therapeutically relevant HtrA inhibitors in the near future. | ||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 6z05.cif.gz | 811.3 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6z05.ent.gz | 653.3 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6z05.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/z0/6z05 ftp://data.pdbj.org/pub/pdb/validation_reports/z0/6z05 | HTTPS FTP |
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-関連構造データ
-リンク
-集合体
登録構造単位 |
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-要素
#1: タンパク質 | 分子量: 47182.379 Da / 分子数: 12 / 由来タイプ: 組換発現 由来: (組換発現) Campylobacter jejuni (カンピロバクター) 遺伝子: htrA, AY595_01940, B7T14_05270, BB943_00930, BB980_02965, BOI95_01210, CA962_01090, CKH46_05385, CW563_00090, DK813_04915, DQX79_02990, DUH84_04555, DW530_01050, F0N90_05545, F1O60_00260, ...遺伝子: htrA, AY595_01940, B7T14_05270, BB943_00930, BB980_02965, BOI95_01210, CA962_01090, CKH46_05385, CW563_00090, DK813_04915, DQX79_02990, DUH84_04555, DW530_01050, F0N90_05545, F1O60_00260, F3L27_00375, F6319_03730, F7858_01545, FR416_00510, FRM19_03410, FRQ83_01860, FZ422_01615, FZU65_03575 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: A0A620LS18 |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
-試料調製
構成要素 | 名称: HtrA dodecamer / タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT |
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分子量 | 実験値: NO |
由来(天然) | 生物種: Campylobacter jejuni (カンピロバクター) |
由来(組換発現) | 生物種: Escherichia coli (大腸菌) |
緩衝液 | pH: 7.4 |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
急速凍結 | 凍結剤: ETHANE |
-電子顕微鏡撮影
顕微鏡 | モデル: TFS GLACIOS |
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電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 200 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD |
撮影 | 電子線照射量: 44 e/Å2 フィルム・検出器のモデル: FEI FALCON III (4k x 4k) |
-解析
CTF補正 | タイプ: NONE |
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対称性 | 点対称性: T (正4面体型対称) |
3次元再構成 | 解像度: 5.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 113843 / 対称性のタイプ: POINT |