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

Abstract

The recent characterization of the prokaryotic Cys2His2 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 Ϸ300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys2His2 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, Ros56-142C82D, 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 Cys2His2 coordination, in Ros homologues can either exploit a CysAspHis2 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.Cys2His2 zinc finger ͉ DNA binding proteins ͉ metal binding proteins ͉ Ros protein I n eukaryotic organisms the abundant Cys 2 His 2 zinc-finger domain, involved in relevant protein-nucleic acid and proteinprotein interactions, consists of Ͻ30 aa and a zinc ion, tetrahedrally coordinated by 2 histidine nitrogens and 2 cysteine sulfurs and essential for stabilizing the ␤␤␣ fold (1-5). In prokaryotic organisms, the first Cys 2 His 2 zinc-finger domain has been identified only recently in the transcriptional regulator Ros from Agrobacterium tumefaciens (6). The Ros zinc-binding domain contains 2 cysteines occupying the first 2 coordinating positions and 3 histidines (His-92, His-96, and His-97; see Fig. 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 ) 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 gl...

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

“…The studies presented here, for instance, demonstrate that mucR expression in B. abortus 2308 is controlled by repressive autoregulation, and studies by Mirabella et al suggest that mucR expression in B. melitensis 16 M may be induced by osmotic stress (45). The results presented in this report also support the contention that the activity of MucR-type transcriptional regulators is dependent upon the availability of divalent cations (22). Given the link between MucR and iron utilization in Brucella, it is possible that iron availability plays a role in the regulation of MucR activity.…”

“…In fact, the origin of the Zn finger motif-containing proteins has been the source of some debate in recent years. Due to the close association of the alphaproteobacteria with eukaryotic host cells, it has been suggested that an ancestral alphaproteobacterium acquired a gene encoding the Zn finger protein from a eukaryotic host (19,20); however, others have proposed that Zn finger proteins are of bacterial origin (21,22). Regardless of their origin, the Zn finger motif-containing proteins, such as Ros and MucR, are essential for the biology of many members of the alphaproteobacteria.…”

NMR assignments of the DNA binding domain of Ml4 protein from Mesorhizobium loti

“…The identity between the Ros homologues includes the amino acids that form the extensive hydrophobic core in Ros while, generally, the Cys 2 His 2 zinc coordination sphere is poorly conserved in these proteins. We have recently demonstrated 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 from Mesorhizobium loti can either or exploit a CysAspHis 2 coordination sphere or lose the metal, while still preserving the DNA-binding activity (Baglivo et al 2009). Among the analyzed Ros homologues from M. loti, Ml4 is a zinc-lacking protein that does not contain the Cys 2 His 2 motif and is able to bind the same Ros DNA target sequence with high affinity (Baglivo et al 2009).…”

“…We have recently demonstrated 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 from Mesorhizobium loti can either or exploit a CysAspHis 2 coordination sphere or lose the metal, while still preserving the DNA-binding activity (Baglivo et al 2009). Among the analyzed Ros homologues from M. loti, Ml4 is a zinc-lacking protein that does not contain the Cys 2 His 2 motif and is able to bind the same Ros DNA target sequence with high affinity (Baglivo et al 2009). Here we report the NMR assignments of the Ml4 protein DNA binding domain (residue 52-151), as an important step toward elucidating at a molecular level how this prokaryotic domain can overcome the metal requirement for proper folding and DNA-binding activity.…”

“…Here we report the NMR assignments of the Ml4 protein DNA binding domain (residue 52-151), as an important step toward elucidating at a molecular level how this prokaryotic domain can overcome the metal requirement for proper folding and DNA-binding activity. (Baglivo et al 2009 (Fig. 1) was recorded with the following parameters: the number of complex points and acquisition times were 256, 160 ms for 15 N (F1), and 2,048, 128 ms for 1 H (F2).…”