Switching Metal Ion Coordination and DNA Recognition in a Tandem CCHHC-type Zinc Finger Peptide

Abstract

Neural Zinc Finger Factor-1 (NZF-1) and Myelin Transcription Factor 1 (MyT1) are two homologous nonclassical zinc finger (ZF) proteins that are involved in the development of the central nervous system (CNS). Both NZF-1 and MyT1 contain multiple ZF domains, each of which contains an absolutely conserved Cys2His2Cys motif. All three cysteines and the second histidine have been shown to coordinate Zn(II); however, the role of the first histidine remains unresolved. Using a functional form of NZF-1 that contains two ZF domains (NZF-1-F2F3), mutant proteins in which each histidine was sequentially mutated to a phenylalanine were prepared to determine the role(s) of the histidine residues in DNA recognition. When the first histidine is mutated, the protein binds Zn(II) in an analogous manner to the native protein. Surprisingly, this mutant does not bind to target DNA (β-RAR), suggesting that the noncoordinating histidine is critical for sequence selective DNA recognition. The first histidine will coordinate Zn(II) when the second histidine is mutated; however, the overall fold of the protein is perturbed leading to abrogation of DNA binding. NZF-1-F2F3 selectively binds to a specific DNA target sequence (from β-RAR) with high affinity (nM); while its homologue MyT1 (MyT1-F2F3), which is 92% identical to NZF-1-F2F3, binds to this same DNA sequence nonspecifically. A single, nonconserved amino acid residue in NZF-1-F2F3 is shown to be responsible for this high affinity DNA binding to β-RAR. When this residue (arginine) is engineered into the MyT1-F2F3 sequence, the affinity for β-RAR DNA increases.

Neural Zinc Finger Factor/Myelin Transcription Factor Proteins: Metal Binding, Fold, and Function

“…Using this method, Wilcox and co-workers determined a dissociation constant in the low nanomolar regime for finger 2 (F2) of MyT1, and this dissociation constant was validated via the Co(II) displacement method. 22 This is in contrast to the midpicomolar regime that was determined using the Co(II) 98 The tighter binding seen for the two-finger construct of MyT1 suggests that there may be cooperative metal binding for these multiple ZF domains. Cooperative metal binding is possible in ZF domains; for example, Seneque and Latour report this behavior in the treble clef CCCC type ZFs.…”

“…Thus, Co(II) is often used as a spectroscopic probe because Co(II) coordinates ZF proteins in a manner similar to that of Zn(II) but has a d 7 electron count resulting in rich spectroscopic properties. 91 98,99 When the second histidine residue is mutated to a glutamine or a phenylalanine, the d−d transitions indicate retention of tetrahedral geometry with a CCHC ligand set ( Figure 4), but the shapes of the d−d transitions differ,…”

“…98,99 This is not observed when the first histidine is mutated to a glutamine or phenylalanine; thus, the mutagenesis/UV−visible spectroscopy studies revealed that the second histidine coordinates metal ions. These results have been validated via the three NMR solution structures: F2 of NZF-1, F5 of MyT1, and F4+F5 of ST18 ( Figure 3D).…”

“…99 However, we reported that when this conserved non-metal-coordinating histidine is mutated to phenylalanine to retain stacking in a two-domain construct of NZF-1 (F2+F3), DNA recognition is abolished, indicating that the role of this histidine is not simply to be involved in π stacking, as equivalent DNA binding should have been observed in the mutant case. 98 Further evidence of a larger role for this nonmetal-coordinating histidine came from sequence analysis. The tyrosine residue thought to be important for π stacking is not conserved in all CCHHC ZF domains.…”

“…24 The lack of α helix and β sheet content observed in the NMR structures of the CCHHC family has been confirmed in circular dichroism (CD) studies of F2+F3 of NZF-1, F2 of MyT1, and F2+F3 of MyT1. 22, 98 In the apo form, the CD is representative of random coil, as evidenced by the large negative signal at 195 nm. Upon addition of Co(II) or Zn(II), this signal becomes less negative and an additional feature appears around 225 nm, indicating the protein has folded but remains largely coiled (Figure 6).…”