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

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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.

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

FEBS J. 2020 Sep 09;:

Authors: Zahn S, Kubatova N, Pyper DJ, Cassidy L, Saxena K, Tholey A, Schwalbe H, Soppa J

Abstract
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). Homologues of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant HVO_2753 grew indistinguishably from the wildtype 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 E. 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 as well as 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.


PMID: 32905660 [PubMed - as supplied by publisher]



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