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j.carres.2014.11.008.pdf | 631.16 kB | Adobe PDF | 見る/開く |
タイトル: | Structural insights into alginate binding by bacterial cell-surface protein. |
著者: | Temtrirath, Kanate Murata, Kousaku Hashimoto, Wataru https://orcid.org/0000-0003-0185-2371 (unconfirmed) |
著者名の別形: | 橋本, 渉 |
キーワード: | Sphingomonas Alginate-binding protein EfeO X-ray crystallography Docking simulation |
発行日: | 2-Mar-2015 |
出版者: | Elsevier Ltd. |
誌名: | Carbohydrate research |
巻: | 404 |
開始ページ: | 39 |
終了ページ: | 45 |
抄録: | A gram-negative Sphingomonas sp. strain A1 inducibly forms a mouth-like pit on the cell surface in the presence of alginate and directly incorporates polymers into the cytoplasm via the pit and ABC transporter. Among the bacterial proteins involved in import of alginate, a cell-surface EfeO-like Algp7 shows an ability to bind alginate, suggesting its contribution to accumulate alginate in the pit. Here, we show identification of its positively charged cluster involved in alginate binding using X-ray crystallography, docking simulation, and site-directed mutagenesis. The tertiary structure of Algp7 was determined at a high resolution (1.99Å) by molecular replacement, although no alginates were included in the structure. Thus, an in silico model of Algp7/oligoalginate was constructed by docking simulation using atomic coordinates of Algp7 and alginate oligosaccharides, where some charged residues were found to be potential candidates for alginate binding. Site-directed mutagenesis was conducted and five purified mutants K68A, K69A, E194A, N221A, and K68A/K69A were subjected to a binding assay. UV absorption difference spectroscopy along with differential scanning fluorimetry analysis indicated that K68A/K69A exhibited a significant reduction in binding affinity with alginate than wild-type Algp7. Based on these data, Lys68/Lys69 residues of Algp7 probably play an important role in binding alginate. |
著作権等: | © 2014 Elsevier Ltd. NOTICE: this is the author's version of a work that was accepted for publication in Carbohydrate Research. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Carbohydrate Research, 404 (2015) doi:10.1016/j.carres.2014.11.008 This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。 |
URI: | http://hdl.handle.net/2433/196076 |
DOI(出版社版): | 10.1016/j.carres.2014.11.008 |
PubMed ID: | 25665777 |
出現コレクション: | 学術雑誌掲載論文等 |
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