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Biological functions, genetic and biochemical characterization, and NMR structure determination of the small zinc finger protein HVO_2753 from Haloferax volcanii
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-09-09 , DOI: 10.1111/febs.15559 Sebastian Zahn 1 , Nina Kubatova 2 , Dennis J Pyper 2 , Liam Cassidy 3 , Krishna Saxena 2 , Andreas Tholey 3 , Harald Schwalbe 2 , Jörg Soppa 1, 4
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-09-09 , DOI: 10.1111/febs.15559 Sebastian Zahn 1 , Nina Kubatova 2 , Dennis J Pyper 2 , Liam Cassidy 3 , Krishna Saxena 2 , Andreas Tholey 3 , Harald Schwalbe 2 , Jörg Soppa 1, 4
Affiliation
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The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one‐domain zinc finger µ‐proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild‐type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in‐depth analysis of a zinc finger µ‐protein in all three domains of life.
中文翻译：
Haloferax volcanii小锌指蛋白HVO_2753的生物学功能，遗传和生化特征以及NMR结构测定
嗜盐古细菌Haloferax volcanii的基因组编码40多个单域锌指µ蛋白。这些中只有一个HVO_2753包含四个C（P）XCG基序，表明存在两个锌结合口袋（ZBP）。HVO_2753的同系物广泛存在于许多euryarchaeota。一个在帧缺失突变体HVO_2753的从野生型不加区别增长在几个媒体，但在蜂拥和在生物膜的形成有一个严重的缺陷。为了进一步分析，该蛋白质在大肠杆菌中同源或异源产生。与许多其他卤代古堡蛋白相比，HVO_2753稳定且在低盐中折叠。只有卤古细菌HVO_2753同源物带有非常亲水的N端，NMR分析表明该区域非常灵活，不是核心结构的一部分。出乎意料的是，尽管存在两个ZBP，NMR分析和荧光测定均显示HVO_2753仅结合一个锌离子。值得注意的是，通过NMR以及在体内对半胱氨酸至丙氨酸突变蛋白的分析互补显示，所有四个C（P）XCG图案对于折叠和功能都是必不可少的。解决了HVO_2753主要构象的NMR溶液结构。出乎意料的是，揭示了ZBP1由C（P）XCG图案1和3组成，ZBP2由C（P）XCG图案2和4组成。与ZBP1相比，有许多迹象表明ZBP2被锌占据。 。据我们所知，这项研究代表了对锌指µ蛋白在生命的所有三个领域中的首次深入分析。
更新日期：2020-09-09
中文翻译：
Haloferax volcanii小锌指蛋白HVO_2753的生物学功能，遗传和生化特征以及NMR结构测定
嗜盐古细菌Haloferax volcanii的基因组编码40多个单域锌指µ蛋白。这些中只有一个HVO_2753包含四个C（P）XCG基序，表明存在两个锌结合口袋（ZBP）。HVO_2753的同系物广泛存在于许多euryarchaeota。一个在帧缺失突变体HVO_2753的从野生型不加区别增长在几个媒体，但在蜂拥和在生物膜的形成有一个严重的缺陷。为了进一步分析，该蛋白质在大肠杆菌中同源或异源产生。与许多其他卤代古堡蛋白相比，HVO_2753稳定且在低盐中折叠。只有卤古细菌HVO_2753同源物带有非常亲水的N端，NMR分析表明该区域非常灵活，不是核心结构的一部分。出乎意料的是，尽管存在两个ZBP，NMR分析和荧光测定均显示HVO_2753仅结合一个锌离子。值得注意的是，通过NMR以及在体内对半胱氨酸至丙氨酸突变蛋白的分析互补显示，所有四个C（P）XCG图案对于折叠和功能都是必不可少的。解决了HVO_2753主要构象的NMR溶液结构。出乎意料的是，揭示了ZBP1由C（P）XCG图案1和3组成，ZBP2由C（P）XCG图案2和4组成。与ZBP1相比，有许多迹象表明ZBP2被锌占据。 。据我们所知，这项研究代表了对锌指µ蛋白在生命的所有三个领域中的首次深入分析。
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