NMR structure verifies the eponymous degenerate 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 ββα 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 motif. The switch in the last ligand maintains the folding topology and hydrophobic core of the classical ZNF motif. CCHC ZNFs are associated with protein-protein interactions but Z* binds DNA. Since the metal chelating site is on the other side of the molecule, it suggests functional preferences are a result of divergent evolution rather than physical constraints on the structure. The structure of Z* provides a context for understanding the domain’s DNA-binding properties and mutations associated with cancers. We expect other ZNFs currently classified as degenerate, are CCHC-type structures like Z*.

[1]  G. Melino,et al.  ZNF750: A Novel Prognostic Biomarker in Metastatic Prostate Cancer , 2023, International journal of molecular sciences.

[2]  Cathy H. Wu,et al.  UniProt: the Universal Protein Knowledgebase in 2023 , 2022, Nucleic acids research.

[3]  E. Callaway.  ‘The entire protein universe’: AI predicts shape of nearly every known protein , 2022, Nature.

[4]  D. Neuhaus.  Zinc finger structure determination by NMR: Why zinc fingers can be a handful , 2022, Progress in nuclear magnetic resonance spectroscopy.

[5]  C. Teschke,et al.  Pulse‐field gradient nuclear magnetic resonance of protein translational diffusion from native to non‐native states , 2022, Protein science : a publication of the Protein Society.

[6]  Xinhan Zhao,et al.  A bioinformatics analysis of zinc finger protein family reveals potential oncogenic biomarkers in breast cancer. , 2022, Gene.

[7]  H. Matsubara,et al.  Biased expression of mutant alleles in cancer-related genes in esophageal squamous cell carcinoma , 2022, Esophagus.

[8]  L. Pan,et al.  Zinc‑finger protein 750 mitigates malignant biological behavior of oral CSC‑like cells enriched from parental CAL‑27 cells , 2021, Oncology Letters.

[9]  O. Wolkenhauer,et al.  Structure–function relationships explain CTCF zinc finger mutation phenotypes in cancer , 2021, Cellular and Molecular Life Sciences.

[10]  K. Kavukcuoglu,et al.  Highly accurate protein structure prediction for the human proteome , 2021, Nature.

[11]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[12]  J. North.  Molecular Genetics of Sebaceous Neoplasia. , 2021, Surgical pathology clinics.

[13]  M. Pellegrini,et al.  IRAK2 has a critical role in promoting feed-forward amplification of epidermal inflammatory responses. , 2021, The Journal of investigative dermatology.

[14]  Jianxiu Yu,et al.  The distinct roles of zinc finger CCHC-type (ZCCHC) superfamily proteins in the regulation of RNA metabolism , 2021, RNA biology.

[15]  Jun Liu,et al.  ZNF750 inhibits the proliferation and invasion of melanoma cells through modulating the Wnt/β-catenin signaling pathway. , 2020, Folia histochemica et cytobiologica.

[16]  I. Amelio,et al.  ZNF750 represses breast cancer invasion via epigenetic control of prometastatic genes , 2020, Oncogene.

[17]  M. Ponce,et al.  Genome-wide analysis of CCHC-type zinc finger (ZCCHC) proteins in yeast, Arabidopsis, and humans , 2020, Cellular and Molecular Life Sciences.

[18]  A. Krężel,et al.  Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains. , 2019, Journal of inorganic biochemistry.

[19]  Hao Yin,et al.  Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9. , 2019, Molecular therapy : the journal of the American Society of Gene Therapy.

[20]  A. Krężel,et al.  Metal binding properties, stability and reactivity of zinc fingers , 2018, Coordination Chemistry Reviews.

[21]  Xiaodong Cheng,et al.  DNA Conformation Induces Adaptable Binding by Tandem Zinc Finger Proteins , 2018, Cell.

[22]  T. Hughes,et al.  The Human Transcription Factors , 2018, Cell.

[23]  Gerry Melino,et al.  Zinc-finger proteins in health and disease , 2017, Cell Death Discovery.

[24]  P. Blackshear,et al.  RNA-binding proteins in immune regulation: a focus on CCCH zinc finger proteins , 2016, Nature Reviews Immunology.

[25]  P. Lavigne,et al.  Solution structure of the 13th C2H2 Zinc Finger of Miz-1. , 2016, Biochemical and biophysical research communications.

[26]  J. Iwahara,et al.  Structural impact of complete CpG methylation within target DNA on specific complex formation of the inducible transcription factor Egr‐1 , 2015, FEBS letters.

[27]  P. Khavari,et al.  ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes , 2014, Genes & development.

[28]  O. Stojadinović,et al.  Epidermal Differentiation in Barrier Maintenance and Wound Healing. , 2014, Advances in wound care.

[29]  Mona Singh,et al.  De novo prediction of DNA-binding specificities for Cys2His2 zinc finger proteins , 2013, Nucleic acids research.

[30]  A. Bax,et al.  Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks , 2013, Journal of Biomolecular NMR.

[31]  Benjamin E. Gross,et al.  Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal , 2013, Science Signaling.

[32]  Dan E. Webster,et al.  Genomic profiling of a human organotypic model of AEC syndrome reveals ZNF750 as an essential downstream target of mutant TP63. , 2012, American journal of human genetics.

[33]  O. Birk,et al.  ZNF750 Is Expressed in Differentiated Keratinocytes and Regulates Epidermal Late Differentiation Genes , 2012, PloS one.

[34]  Z. Siprashvili,et al.  ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation. , 2012, Developmental cell.

[35]  P. Kwok,et al.  Association analysis identifies ZNF750 regulatory variants in psoriasis , 2011, BMC Medical Genetics.

[36]  S. Michel,et al.  Cysteine and histidine shuffling: mixing and matching cysteine and histidine residues in zinc finger proteins to afford different folds and function. , 2011, Dalton transactions.

[37]  S. Futaki,et al.  Metal‐Stimulated Regulation of Transcription by an Artificial Zinc‐Finger Protein , 2010, Chembiochem : a European journal of chemical biology.

[38]  Aaron Klug,et al.  The discovery of zinc fingers and their applications in gene regulation and genome manipulation. , 2010, Annual review of biochemistry.

[39]  Tim J Stevens,et al.  DANGLE: A Bayesian inferential method for predicting protein backbone dihedral angles and secondary structure. , 2010, Journal of magnetic resonance.

[40]  P. Park.  ChIP–seq: advantages and challenges of a maturing technology , 2009, Nature Reviews Genetics.

[41]  R. Emerson,et al.  Adaptive Evolution in Zinc Finger Transcription Factors , 2009, PLoS genetics.

[42]  Y. Chien,et al.  A promoter sequence variant of ZNF750 is linked with familial psoriasis. , 2008, The Journal of investigative dermatology.

[43]  M. Delepierre,et al.  Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation. , 2008, Journal of molecular biology.

[44]  E. Ivanova,et al.  Zinc binding of Tim10: Evidence for existence of an unstructured binding intermediate for a zinc finger protein , 2008, Proteins.

[45]  Donald Kennedy,et al.  Breakthrough of the Year , 2007, Science.

[46]  D. Geiger,et al.  Seborrhea-like dermatitis with psoriasiform elements caused by a mutation in ZNF750, encoding a putative C2H2 zinc finger protein , 2006, Nature Genetics.

[47]  Wayne Boucher,et al.  The CCPN data model for NMR spectroscopy: Development of a software pipeline , 2005, Proteins.

[48]  J. Ladbury,et al.  Solvation and the hidden thermodynamics of a zinc finger probed by nonstandard repair of a protein crevice , 2004, Protein science : a publication of the Protein Society.

[49]  A. Alexandrescu,et al.  NMR structure of the C-terminal domain of SecA in the free state. , 2004, Biochimica et biophysica acta.

[50]  J. Mackay,et al.  CCHX Zinc Finger Derivatives Retain the Ability to Bind Zn(II) and Mediate Protein-DNA Interactions* , 2003, Journal of Biological Chemistry.

[51]  Terry Gaasterland,et al.  Systematic characterization of the zinc-finger-containing proteins in the mouse transcriptome. , 2003, Genome research.

[52]  N. Grishin,et al.  Structural classification of zinc fingers: survey and summary. , 2003, Nucleic acids research.

[53]  J. Mackay,et al.  A class of zinc fingers involved in protein-protein interactions biophysical characterization of CCHC fingers from fog and U-shaped. , 2000, European journal of biochemistry.

[54]  B. Roques,et al.  Structural investigation on the requirement of CCHH zinc finger type in nucleocapsid protein of human immunodeficiency virus 1. , 1999, Biochemistry.

[55]  M J Sippl,et al.  Who solved the protein folding problem? , 1999, Structure.

[56]  A. Jackson,et al.  The mutation rate and cancer. , 1998, Genetics.

[57]  G. Marius Clore,et al.  The solution structure of a specific GAGA factor–DNA complex reveals a modular binding mode , 1997, Nature Structural Biology.

[58]  J. Berg,et al.  The Galvanization of Biology: A Growing Appreciation for the Roles of Zinc , 1996, Science.

[59]  P. Wright,et al.  Zinc is required for folding and binding of a single zinc finger to DNA , 1991, FEBS letters.

[60]  A Klug,et al.  Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes. , 1985, The EMBO journal.

[61]  Lena Jaeger,et al.  Introduction To Protein Structure , 2016 .

[62]  Yang Shen,et al.  Protein structural information derived from NMR chemical shift with the neural network program TALOS-N. , 2015, Methods in molecular biology.

[63]  P. Khavari,et al.  Genetic pathways in disorders of epidermal differentiation. , 2013, Trends in genetics : TIG.

[64]  Charles D Schwieters,et al.  The Xplor-NIH NMR molecular structure determination package. , 2003, Journal of magnetic resonance.

[65]  C. Pabo,et al.  DNA recognition by Cys2His2 zinc finger proteins. , 2000, Annual review of biophysics and biomolecular structure.

[66]  P E Wright,et al.  Use of chemical shifts and coupling constants in nuclear magnetic resonance structural studies on peptides and proteins. , 1994, Methods in enzymology.

[67]  J M Walker,et al.  The bicinchoninic acid (BCA) assay for protein quantitation. , 1994, Methods in molecular biology.

[68]  L. Hood,et al.  Spectroscopic studies of wild-type and mutant "zinc finger" peptides: determinants of domain folding and structure. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[69]  J. Berg,et al.  Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. , 1988, Proceedings of the National Academy of Sciences of the United States of America.