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

It is increasingly recognized that very small proteins (mu-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 mu-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 mu-proteins HVO_0758 and HVO_2753 showed many differences. Taken together, two zinc finger mu-proteins of H. volcanii have been characterized intensively, which emerged as pivotal contributors to swarming behavior and biofilm formation.

Automated summary

It is increasingly recognized that very small proteins, called mu-proteins, are ubiquitously found in all species of the three domains of life, and they fulfill important functions. Through in-depth analysis of two C(P)XCG proteins in Haloferax volcanii, it was found that they play pivotal roles in the swarming behavior and biofilm formation of H. volcanii.