Coordination and oxidation properties of ZBTB20: CX2CX12HX3H-type zinc fingers

Abstract

Zinc finger (ZF) proteins require zinc ion (Zn²⁺) to maintain structural integrity, which facilitates nucleic acid recognition. ZBTB20 is a Broad-complex, Tramtrack, and Bric-à-brac (BTB)–ZF transcription factor that regulates neuronal differentiation in the developing cortex, although the biochemical properties of the ZF domains in ZBTB20 remain to be further investigated. In this study, we demonstrate structural and functional aspects of five CX₂CX₁₂HX₃H-type ZF domains based on the metal dependence of ZBTB20. Circular dichroism (CD) studies indicate that Zn²⁺ preserves the ββα secondary structure of ZBTB20(ZFs), whereas Zn²⁺ removal disrupts this structural motif, indicating the requirement for intact metal–coordination in maintaining the native fold. The d–d transition spectra establish tetrahedral (T_d) metal–coordination and reveal the high oxidative susceptibility of these domains compared with other classical CX₂CX₁₂HX₃H-type ZF proteins. Interaction studies of ZBTB20(ZFs) identify brn2-F3 (K_d = 1.2 ± 0.1 nM) to represent the specific binding region within the human brn2 promoter, which provides a mechanistic explanation for ZBTB20-mediated Brain-2 (Brn2) repression in neuronal development. Reconstruction of apo-ZBTB20(ZFs) with metal ions result in diminished binding to brn2-F3, highlighting that strict preservation of native Zn²⁺–coordination is essential for recognition of nucleic acids. Overall, our work advances new insights into the Zn²⁺ dependent behavior of the ZBTB family and establishes a biochemical basis for selective promoter recognition.