Biological functions, genetic and biochemical characterization, and NMR structure determination of the small zinc finger protein HVO_2753 from Haloferax volcanii

  • 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.

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Author:Sebastian Zahn, Nina Kubatova, Dennis J. Pyper, Liam Cassidy, Krishna SaxenaORCiDGND, Andreas Tholey, Harald SchwalbeORCiDGND, Jörg SoppaORCiD
URN:urn:nbn:de:hebis:30:3-638531
DOI:https://doi.org/10.1111/febs.15559
ISSN:1439-7633
Parent Title (English):The FEBS journal
Publisher:Wiley-Blackwell
Place of publication:Oxford [u.a.]
Document Type:Article
Language:English
Date of Publication (online):2020/09/09
Date of first Publication:2020/09/09
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2022/03/09
Tag:Haloferax volcanii; NMR solution structure; small protein; zinc finger; µ-protein
Volume:288
Issue:6
Page Number:21
First Page:2042
Last Page:2062
Note:
The collaborative project was funded by the German Research Council (Deutsche Forschungsgemeinschaft, DFG) in the framework of the Priority Program SPP2002 'Small proteins in prokaryotes' (www.spp2002.uni-kiel.de). The projects of the groups of JS and HS and the Z1 project headed by AT were involved, and the project numbers are JS264/26-1, SCHW 701/21-1, and TH872/10-1. Open access funding enabled and organized by ProjektDEAL.
HeBIS-PPN:494118164
Institutes:Biochemie, Chemie und Pharmazie
Biowissenschaften
Wissenschaftliche Zentren und koordinierte Programme / Zentrum für Biomolekulare Magnetische Resonanz (BMRZ)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Licence (German):License LogoCreative Commons - Namensnennung 4.0