A Novel Type of Zinc Finger DNA Binding Domain in the Agrobacterium tumefaciens Transcriptional Regulator Ros

Abstract

Transcriptional factors bearing a Cys(2)His(2) zinc finger were thought to be confined to eukaryotes, but recent studies have suggested their presence also in prokaryotes. In this paper, we report the first complete functional characterization of the DNA binding domain present in the putative Cys(2)His(2) zinc finger-containing prokaryotic transcriptional regulator Ros from Agrobacterium tumefaciens. We demonstrate that in the single zinc binding motif present in the Ros protein the metal ion is coordinated by two cysteines (Cys79 and Cys82) and two histidines (His92 and His97), separated by a shorter spacer with respect to the eukaryotic classical Cys(2)His(2) domains. The Cys(2)His(2) zinc finger motif is essential for Ros DNA binding and is part of a larger DNA binding domain which includes four basic regions located on either side of the finger, one at the N-terminus and three at the C-terminus. The one described here is a novel type of DNA binding domain containing a noncanonical Cys(2)His(2) zinc finger motif which, by sequence alignment, seems to be conserved in all the bacterial putative zinc finger proteins identified so far. Interestingly, basic amino acids have been shown to be important in stabilizing the DNA binding of eukaryotic single Cys(2)His(2) zinc finger domains, confirming that the modality of DNA binding using a single zinc finger motif flanked by basic residues is widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic, even if in each kingdom it presents its peculiarity.

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

“…1) is substituted by an aspartic acid. Ros 56 -142 C82D has been tested by EMSA using the VirC oligonucleotide recognised by the Ros protein (7) or an oligonucleotide with an unrelated sequence (NS), as probes ( Fig. 2A, lanes 1 and 2).…”

“…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.…”

“…1). 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).…”

“…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.…”

“…In particular, Ros 56 -142 globular domain consists of 58 aa, arranged in a ␤␤␤␣␣ topology and stabilized by an extensive 15-residue hydrophobic core. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated to be essential for Ros DNA binding (7). In recent years, Ͼ300 Ros homologues have been identified in a large number of bacteria, mostly belonging to the ␣ subdivision of proteobacteria (9,10).…”