The prokaryotic Cys ₂ His ₂ zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain

Abstract

The first putative prokaryotic Cys2His2 zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys2His2 zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys2His2 zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys2His2 zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9 -66), is arranged in a ␤␤␤␣␣ topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15 N R1, 15 N R2, and heteronuclear 15 N-{ 1 H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain.DNA binding proteins ͉ NMR spectroscopy ͉ Ros protein

The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains

“…On the basis of the Chemical Shift Index (19) we derived a secondary structure prediction, shown in Fig. 5, which is essentially identical to that obtained for Ros and consistent with its high-resolution structure (8). This finding, together with the observation that the amino acids involved in the extensive Ros hydrophobic core are highly conserved ( Fig.…”

“…The protein zinc content has been analyzed by inductively coupled plasma mass spectrometry (ICP-MS), demonstrating a ratio between the The positions corresponding to Ros His-96 and His-97 residues, able to function alternatively as the fourth coordinating position in Ros (7), are both indicated. In bold are indicated the amino acids constituting the Ros hydrophobic core (8). The basic residues shown to be important for Ros DNA binding (7) are underlined.…”

“…Along with the functional and structural characterization of the prokaryotic zinc finger domain (6)(7)(8) this study allows us to formulate a comparison between the main features of zinc-finger domains in prokaryota and eukaryota. Definitely, the 2 domains have a very similar zinc coordination sphere (8) arranged in a ␤␤␣ motif, but their global folds are significantly different.…”

“…We (7) demonstrated that in the Ros protein the histidines involved in zinc coordination are His-92, acting as the third coordinating residue, and His-97 as the fourth; when His-97 is mutated in Ala, the protein is still able to bind zinc and DNA with His-96 acting as the fourth coordinating residue. The NMR structure of Ros DNA-binding domain (Ros 56 -142 ) has shown that the prokaryotic Cys 2 His 2 zinc-finger domain, although having a similar zinc coordination sphere, possesses a novel protein fold, which is very different from that of the eukaryotic counterpart (8). In particular, Ros 56 -142 globular domain consists of 58 aa, arranged in a ␤␤␤␣␣ topology and stabilized by an extensive 15-residue hydrophobic core.…”

“…Interestingly, our extended database analysis of Ros homologous sequences confirms the first observation reported by Moreira and Rodriguez-Valera (10) that the 4 zinc-coordinating residues in Ros are all together poorly preserved with only the first coordinating histidine conserved in almost all of the Ros homologues. In particular, the first cysteine is replaced by a serine in Ϸ40% of the homologues and the second cysteine is replaced by an aspartate in Ϸ60%; the 2 possible positions for the fourth coordinating residue, which in Ros are occupied by the 2 adjacent histidines (7,8), in 50% of the homologues are both replaced by different pairs of amino acids, such as arginineglycine, glutamine-tyrosine, lysine-tyrosine, leucine-glycine, and others. Such a high variability in the putative coordination sphere raises the question of whether the zinc ion is present in all of the Ros homologues.…”