Zinc-induced folding and solution structure of the eponymous novel zinc finger from the ZC4H2 protein

ABSTRACT The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil, and a single novel zinc finger with four cysteines and two histidines from which the protein gets its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using a synthetic peptide fragment that the zinc finger of ZC4H2 is genuine, and folds around zinc ion with picomolar affinity. NMR pH titration of histidines and UV-Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger likely perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative unprecedented functions. Competing Interest Statement The authors have declared no competing interest. Footnotes ABBREVIATIONS: AF3, Alpha Fold 3; CD, circular dichroism; DOSY, diffusion ordered spectroscopy; DSS, 3-(Trimethylsilyl)propane-1-sulfonic acid; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; HSQC, heteronuclear single quantum correlation; LMCT, ligand-to-metal charge transfer; NLS, nuclear localization signal; NMR, nuclear magnetic resonance, NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; PDB, protein data bank; pLDDT, predicted local distance difference test; RMSD, root mean square deviation; SD, standard deviation; TCEP, tris(2-carboxyethyl)phosphine; TOCSY, total correlation spectroscopy; UV-Vis, ultraviolet-visible spectrophotometry; ZARD, ZC4H2-associated rare disorders; ZC4H2-ZL, long fragment comprised of residues 186-212 of the ZC4H2 protein; ZC4H2-ZS, short fragment comprised of residues 188-207 of the ZC4H2 protein; ZnF, zinc finger.