Characterization of the zinc finger μ-protein HVO_0758 from Haloferax volcanii: biological roles, zinc binding, and NMR solution structure

Deniz Üresin; Dennis J Pyper; Andreas Borst; Lydia Hadjeras; Rick Gelhausen; Rolf Backofen; Cynthia Sharma; Harald Schwalbe; Jörg Soppa

doi:10.3389/fmicb.2023.1280972

Characterization of the zinc finger μ-protein HVO_0758 from Haloferax volcanii: biological roles, zinc binding, and NMR solution structure

Front Microbiol. 2023 Nov 29:14:1280972. doi: 10.3389/fmicb.2023.1280972. eCollection 2023.

Authors

Affiliations

¹ Institute for Molecular Biosciences, Goethe University, Frankfurt, Germany.
² Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University, Frankfurt, Germany.
³ Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
⁴ Bioinformatics Group, Department of Computer Science, University of Freiburg, Freiburg, Germany.
⁵ Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany.

^# Contributed equally.

Abstract

It is increasingly recognized that very small proteins (μ-proteins) are ubiquitously found in all species of the three domains of life, and that they fulfill important functions. The halophilic archaeon Haloferax volcanii contains 282 μ-proteins of less than 70 amino acids. Notably, 43 of these contain two C(P)XCG motifs, suggesting their potential to complex a zinc ion. To explore the significance of these proteins, 16 genes encoding C(P)XCG proteins had been deleted, and the majority of mutants exhibited phenotypic differences to the wild-type. One such protein, HVO_2753, was thoroughly characterized in a previous study. In the present study an in-depth analysis of a second protein, HVO_0758, was performed. To achieve this goal, the HVO_0758 protein was produced heterologously in Escherichia coli and homologously in H. volcanii. The purified protein was characterized using various biochemical approaches and NMR spectroscopy. The findings demonstrated that HVO_0758 is indeed a bona fide zinc finger protein, and that all four cysteine residues are essential for folding. The NMR solution structure was solved, revealing that HVO_0758 is comprised of an N-terminal alpha helix containing several positively charged residues and a globular core with the zinc finger domain. The transcriptomes of the HVO_0758 deletion mutant and, for comparison, the HVO_2753 deletion mutant were analyzed with RNA-Seq and compared against that of the wild-type. In both mutants many motility and chemotaxis genes were down-regulated, in agreement to the phenotype of the deletion mutants, which had a swarming deficit. The two H. volcanii zinc-finger μ-proteins HVO_0758 and HVO_2753 showed many differences. Taken together, two zinc finger μ-proteins of H. volcanii have been characterized intensively, which emerged as pivotal contributors to swarming behavior and biofilm formation.

Keywords: Archaea; Haloferax volcanii; NMR solution structure; RNA-Seq; TALON; microproteins; small proteins; zinc finger.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This collaborative work was supported by the German Research Foundation (DFG) in the framework of priority program SPP2002 “Small Proteins in Prokaryotes, an Unexplored World” (grant SO 264/26 to JS, grant SH580/7-1 and SH580/7-2 to CMS, grant BA 2168/21-2 to RB, grant BE 3869/5-1; www.spp2002.uni-kiel.de/wordpress). The center for biomolecular magnetic resonance (BMRZ) is supported by the state of Hesse, Germany.