Solution Structure of the Novel CH-domain zinc finger from the puberty regulator Makorin-3

The makorin (MKRN) family of E3 ubiquitin ligases (MKRN1–4) regulates diverse biological processes, including reproduction, neurogenesis, and immune function. The first identified member, MKRN3, is an inhibitor of sexual development that is the site of inherited mutations linked to central precocious puberty (CPP). All makorin proteins share a distinctive cysteine/histidine-rich (CH) domain that has not been previously characterized experimentally. In MKRN3, the CH domain lies between the second C3H zinc finger and the RING domain. The C3H(2)–CH–RING segment appears particularly sensitive to CPP mutations suggesting it may constitute a structure-function unit. Using CD and NMR spectroscopy, we show that the CH domain folds upon coordination of a single Zn 2+ ion with picomolar affinity. Spectroscopic and NMR pH-titration analyses identify a CCHC-type metal-binding site, typical of zinc fingers with protein-interaction functions. The NMR structure reveals the CH-domain adopts a canonical ββα zinc finger fold, despite atypical ligand spacing and the absence of conserved hydrophobic residues that usually stabilize this type of motif. Thermal denaturation monitored by multiple spectroscopic probes indicates sequential unfolding, with side-chain packing disrupted near 33 °C but zinc-stabilized secondary structure persisting to ∼63 °C, consistent with a molten-globule intermediate at high temperature. The function of the CH-domain remains unknown, but it could play a role in allosterically transmitting information on the RNA-bound state of the preceding C3H(2) domain to the subsequent RING domain. Based on a similar metal ligand spacing to a zinc finger from the protein FAAP20 and AlphaFold modeling, the CH-domain may have a ubiquitin-binding function, but this will need to be verified experimentally as AlphaFold also confidently predicts complexes with unrelated random proteins.