NMR structure verifies the eponymous zinc finger domain of transcription factor ZNF750.

ZNF750 is a nuclear transcription factor that activates skin differentiation and has tumor suppressor roles in several cancers. Unusually, ZNF750 has only a single zinc-finger (ZNF) domain, Z*, with an amino acid sequence that differs markedly from the CCHH family consensus. Because of its sequence differences Z* is classified as degenerate, presumed to have lost the ability to bind the zinc ion required for folding. AlphaFold predicts an irregular structure for Z* with low confidence. Low confidence predictions are often inferred to be intrinsically disordered regions of proteins, which would be the case if Z* did not bind Zn2+. We use NMR and CD spectroscopy to show that a 25-51 segment of ZNF750 corresponding to the Z* domain folds into a well-defined antiparallel ßßα tertiary structure with a pM dissociation constant for Zn2+ and a thermal stability >80 °C. Of three alternative Zn2+ ligand sets, Z* uses a CCHC rather than the expected CCHH ligating motif. The switch in the last ligand maintains the folding topology and hydrophobic core of the classical ZNF motif. CCHC ZNFs are typically associated with protein-protein interactions, raising the possibility that ZNF750 interacts with DNA through other proteins rather than directly. The structure of Z* provides context for understanding the function of the domain and its cancer-associated mutations. We expect other ZNFs currently classified as degenerate could be CCHC-type structures like Z*.

Pulse-field gradient nuclear magnetic resonance of protein translational diffusion from native to non-native states.

Hydrodynamic radii (Rh -values) calculated from diffusion coefficients measured by pulse-field-gradient nuclear magnetic resonance are compared for folded and unfolded proteins. For native globular proteins, the Rh -values increase as a power of 0.35 with molecular size, close to the scaling factor of 0.33 predicted from polymer theory. Unfolded proteins were studied under four sets of conditions: in the absence of denaturants, in the presence of 6 M urea, in 95% dimethyl sulfoxide (DMSO), and in 40% hexafluoroisopropanol (HFIP). Scaling factors under all four unfolding conditions are similar (0.49-0.53) approaching the theoretical value of 0.60 for a fully unfolded random coil. Persistence lengths are also similar, except smaller in 95% DMSO, suggesting that the polypeptides are more disordered on a local scale with this solvent. Three of the proteins in our unfolded set have an asymmetric sequence-distribution of charged residues. While these proteins behave normally in water and 6 M urea, they give atypically low Rh -values in 40% HFIP and 95% DMSO suggesting they are forming electrostatic hairpins, favored by their asymmetric sequence charge distribution and the low dielectric constants of DMSO and HFIP. While diffusion-ordered NMR spectroscopy can separate small molecules, we show a number of factors combine to make protein-sized molecules much more difficult to resolve in mixtures. Finally, we look at the temperature dependence of apparent diffusion coefficients. Small molecules show a linear temperature response, while large proteins show abnormally large apparent diffusion coefficients at high temperatures due to convection, suggesting diffusion reference standards are only useful near 25°C.

NMR Mapping of Disordered Segments from a Viral Scaffolding Protein Enclosed in a 23 MDa Procapsid.

Scaffolding proteins (SPs) are required for the capsid shell assembly of many tailed double-stranded DNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, only one high-resolution structure is available for SPs within procapsids. Here, we use the inherent size limit of NMR to identify mobile segments of the 303-residue phage P22 SP free in solution and when incorporated into a ∼23 MDa procapsid complex. Free SP gives NMR signals from its acidic N-terminus (residues 1-40) and basic C-terminus (residues 264-303), whereas NMR signals from the middle segment (residues 41-263) are missing because of intermediate conformational exchange on the NMR chemical shift timescale. When SP is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix domain disappear because of binding to the procapsid interior. Signals from the N-terminal domain persist, indicating that this segment retains flexibility when bound to procapsids. The unstructured character of the N-terminus, coupled with its high content of negative charges, is likely important for dissociation and release of SP during the double-stranded DNA genome packaging step accompanying phage maturation.