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

Abstract

The recent characterization of the prokaryotic Cys(2)His(2) zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified approximately 300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys(2)His(2) zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros(56-142)C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys(2)His(2) coordination, in Ros homologues can either exploit a CysAspHis(2) coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.

Fig. 1.

Sequence alignment of the 10 Ros homologues identified in the M. loti genome. The accession numbers of the proteins that have been analyzed are in italics, and the names used in this study are in parentheses. The arrows and the boxes indicate the positions corresponding to the coordination residues in Ros. 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. The asterisk indicates a conserved residues; the column indicates a conservative substitution; a period indicates a semiconservative substitution.

Fig. 2.

EMSA of protein DNA binding. The proteins used are indicated; residues corresponding to the coordinating positions in the Ros protein are in parentheses, and the residues occupying the 2 possible fourth coordinating positions are in italics. DNA-binding specificity has been demonstrated by incubating the proteins with the labeled VirC oligonucleotide (VirC, lanes 1) or a labeled oligonucleotide with an unrelated sequence (NS, lanes 2). EDTA (50 mM) has been added to Ros_56–142C82D and Ml1_53–149 (lanes 3) to investigate the zinc requirement for DNA binding.

Fig. 3.

Definition of the role of the His residues in zinc coordination by NMR analysis in Ros_56–142C82D. A portion of the ¹H-¹⁵N HSQC J18 spectrum of Ros_56–142C82D acquired at pH 6.8 is shown. The gray rectangles indicate the nitrogen chemical-shift ranges typical for His involved in zinc ion coordination.

Fig. 4.

Definition of the role of the His residues in zinc coordination by NMR analysis in Ml4_56–151, Ml5_56–154, and Ml5_56–154 S77C. Portions of the ¹H-¹⁵N HSQC J18 spectra acquired at pH 6.8 are shown. The proteins analyzed are indicated. The gray rectangles indicate the nitrogen chemical-shift ranges typical for His involved in zinc ion coordination. (A) The single His present in Ml4_56–151 is a N_ε2-H tautomer and does not coordinate Zn(II). (B and C) Upon mutation of Ser-77 to Cys in Ml5_56–154, the 2 His residues change their WT N_ε2-H tautomeric forms (B) to N_δ1-H tautomeric forms and their nitrogen chemical shifts fall in the typical regions of chemical shifts for His involved in zinc ion coordination (C).

Fig. 5.

Secondary structure elements (β-strands and helices) in Ml4_56–151 sequence as derived by the Chemical Shift Index, based on H_α, C_α and C_β resonance assignments.