Zinc:DNA‐Binding Proteins

Abstract

Zinc‐containing nucleic acid‐binding proteins are ubiquitous in nature and interact with all types of nucleic acids, including ssDNA, duplex DNA, and RNA. The primary role played by these Zn(II) coordination complexes is to provide local or global stability to these proteins that function in transcriptional regulation, DNA metabolism, and nucleic acid packaging. These proteins are now broadly designated as zinc finger (zf) proteins. Six structural classes of zf proteins are discussed here that are distinguished from one another on the basis of the secondary and tertiary structure of the zf domain, the nature of the ligating side chains (Cys and/or His), and the linear spacing of amino acid ligands in the primary structure. These structural zinc sites are compared with metalloregulatory zinc coordination complexes found in metal‐sensing transcriptional regulatory proteins that control zinc homeostasis in bacteria (e.g. E. coli ZurR and ZntR; S. aureus CzrA; Synechococcus SmtB), lower eukaryotes ( S. cerevisiae Zap1), and mammalian cells (MTF‐1). Structural and functional comparisons of the bacterial zinc sensors with homologous sensors specific for other transition metal ions (e.g. Cu, Ni, Mn) and xenobiotics (e.g. Cd, Pb, and Hg) suggest that coordination number and therefore geometry are primary determinants of metal selectivity of metal sensor proteins in vivo.

Structural Insights into Homo- and Heterotropic Allosteric Coupling in the Zinc Sensor S. aureus CzrA from Covalently Fused Dimers

“…The free energy of allosteric coupling (Δ G c ) of Zn(II) and DNA binding by fCzrAs can be estimated from the following (eq 1): where Δ G 1 c is operationally defined as deriving from the first fCzrA-CzrO binding event only (i.e., K 1 ) since higher order complexes could not be observed for Zn(II)-saturated fCzrAs (Figure ). Δ G 1 c for the wt -CzrA and CzrA(Δ3−9) homodimers are indistinguishable, 6.3 (±0.2) and 6.5 (±0.2) kcal mol -1 (Figure S4, Supporting Information), indicative of strong heterotropic allosteric negative coupling for the unfused dimers ).…”

“…To directly test this proposal, we exploited the fused dimer system to introduce a single poorly metal liganding substitution into one of the two metal sites independently in the context of 10L-fCzrA . Two 10L-fCzrA substitutions were prepared.…”

“…These are denoted H97N 10L-fCzrA, which introduces the H97N mutation into the N-terminal protomer domain and perturbs metal site 2, closest to the peptide linker (see Figure B), and H197N 10L-fCzrA, which introduces the same substitution into the C-terminal protomer domain perturbing metal site 1, distant from the linker (see Figure B). An Asn substitution of His97 was chosen because previous work has shown that the H97N substitution in the context of the CzrA homodimer abrogates allosteric coupling (Δ G c = 0), and these metal sites adopt non-native coordination geometries and bind Zn(II) weakly ( K Zn1 = 6.8 (±0.3) × 10 6 M -1 ; K Zn2 ≤ 1 × 10 5 M -1 ) relative to wt -CzrA . Thus, each substitution is expected to selectively disrupt just one of the His97-mediated H-bonding pathways linked to allostery since the critical H ε2 proton is obviously not present in this mutant …”