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

Rua, Antonio J.; Whitehead, R.; Alexandrescu, Andrei T.

doi:10.1016/j.yjsbx.2023.100093

Cited by 3 publications

(16 citation statements)

References 74 publications

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“…There is an increasingly prevalent view in modern biology that protein structures can be largely predicted by homology to the large number of known structures. The mis-annotation of the Z7 domain described in this work, our recent correction of the degenerate annotation for the Z* domain of Z750 (Rua et al 2023), as well as several annotations we corrected previously (Rua et al 2023) or are currently working on correcting, suggest there is still much to learn about the biophysical and structural properties of the resilient and versatile zinc-finger fold.…”

Section: Discussionmentioning

confidence: 81%

“…Note that the AlphaFold algorithm (Jumper et al 2021) used by ColabFold does not consider cofactors, so the Zn 2+ ion is not modeled in the structure. In contrast to the Z* domain from ZNF750 we recently described where AlphaFold models the structure incorrectly (Rua et al 2023), the ColabFold model for Z7 is very close to the NMR structure giving a backbone RMSD of 1.6 Å (Table 1). Like in the NMR structure, H26 in the ColabFold model is too far to be involved in the Zn 2+ coordination site (Fig.…”

Section: The Z7 Domain Has a Classical Znf Bba Foldmentioning

confidence: 75%

“…1C). We used CD spectroscopy to obtain information on Zn 2+ avidity in a competition assay with EGTA (Rua et al 2023). For Mut2 we used the CD signal at 222 nm to follow the Zn 2+ titration (Fig 4B ), whereas 220 nm was used for WT, and 214 nm for Mut1.…”

Section: Dissociation Constants For Zn 2+ and Co 2+ Are Similar For W...mentioning

confidence: 99%

“…The folding status of these degenerate ZNFs is currently not easy to predict. We recently reported the NMR structure of the single ZNF domain of ZNF750 (Rua et al 2023). Neither the UniProt database nor AlphaFold were able to correctly establish the folding status of the domain or predict its structure.…”

Section: Introductionmentioning

confidence: 99%

“…Neither the UniProt database nor AlphaFold were able to correctly establish the folding status of the domain or predict its structure. We corrected examples of false degenerate annotations for three other domains (ZFAT-Z14, ZNF32-Z7, TZAP-Z2) for which there were experimental structures in the Protein Databank (PDB codes 2RV1, 2EPC, 2DLQ), and suggested several domains annotated as degenerate are probably functional CCHC-type ZNFs (Rua et al 2023). More recently we initiated NMR studies of a further five ZNF domains that despite being either annotated as degenerate or not being recognized as ZNFs by UniProt, adopt folded tertiary structure in the presence of Zn 2+ (A.T.A.…”

Section: Introductionmentioning

confidence: 99%

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Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Rua,

Alexandrescu

2024

Preprint

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Domain Z7 of nuclear transcription factor ZNF711 has the consensus last metal-ligand H23 found in odd-numbered zinc-fingers of this protein replaced by a phenylalanine. Ever since the discovery of ZNF711 it has been thought that Z7 is probably non-functional because of the H23F substitution. The presence of H26 three positions downstream prompted us to examine if this histidine could substitute as the last metal ligand. The Z7 domain adopts a stable tertiary structure upon metal binding. The NMR structure of Zn2+-bound Z7 shows the classical ββα-fold of CCHH zinc fingers. Mutagenesis and pH titration experiments indicate that H26 is not involved in metal binding and that Z7 has a tridentate metal-binding site comprised of only residues C3, C6, and H19. By contrast, an F23H mutation that introduces a histidine in the consensus position forms a tetradentate ligand. The structure of the WT Z7 is stable causing restricted ring-flipping of phenyalanines 10 and 23. Dynamics are increased with either the H26A or F23H substitutions and aromatic ring rotation is no longer hindered in the two mutants. The mutations have only small effects on the Kd values for Zn2+and Co2+and retain the high thermal stability of the WT domain above 80oC. Like two previously reported designed zinc fingers with the last ligand replaced by water, the WT Z7 domain is catalytically active, hydrolyzing 4-nitophenyl acetate. We discuss the implications of naturally occurring tridentate zinc fingers for cancer mutations and drug targeting of notoriously undruggable transcription factors. Our findings that Z7 can fold with only a subset of three metal ligands suggests the recent view that most everything about protein structure can be predicted through homology modeling might be premature for at least the resilient and versatile zinc-finger motif.

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Section: Discussionmentioning

confidence: 81%

Section: The Z7 Domain Has a Classical Znf Bba Foldmentioning

confidence: 75%

Section: Dissociation Constants For Zn 2+ and Co 2+ Are Similar For W...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Rua,

Alexandrescu

2024

Preprint

Self Cite

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Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Rua,

Alexandrescu

2024

Protein Science

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Domain Z7 of nuclear transcription factor ZNF711 has the consensus last metal‐ligand H23 found in odd‐numbered zinc fingers of this protein replaced by a phenylalanine. Ever since the discovery of ZNF711, it has been thought that Z7 is probably non‐functional because of the H23F substitution. The presence of H26 three positions downstream prompted us to examine if this histidine could substitute as the last metal‐ligand. The Z7 domain adopts a stable tertiary structure upon metal‐binding. The NMR structure of Zn2+‐bound Z7 shows the classical ββα‐fold of CCHH zinc fingers. Mutagenesis and pH titration experiments indicate that H26 is not involved in metal binding and that Z7 has a tridentate metal‐binding site comprised of only residues C3, C6, and H19. By contrast, an F23H mutation that introduces a histidine in the consensus position forms a tetradentate ligand. The structure of the WT Z7 is stable causing restricted ring‐flipping of phenylalanines 10 and 23. Dynamics are increased with either the H26A or F23H substitutions and aromatic ring rotation is no longer hindered in the two mutants. The mutations have only small effects on the Kd values for Zn2+ and Co2+ and retain the high thermal stability of the WT domain above 80°C. Like two previously reported designed zinc fingers with the last ligand replaced by water, the WT Z7 domain is catalytically active, hydrolyzing 4‐nitrophenyl acetate. We discuss the implications of naturally occurring tridentate zinc fingers for cancer mutations and drug targeting of notoriously undruggable transcription factors.

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Solution structure of the Z0 domain from transcription repressor BCL11A sheds light on the sequence properties of protein-binding zinc-fingers

Harris,

Whitehead,

Alexandrescu

2024

Preprint

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The transcription repressor BCL11A, which governs the switch from fetal to adult hemoglobin during development, is the target of the first FDA-approved CRISPR/Cas9 gene-editing therapy in humans. By targeting BCL11A, fetal hemoglobin expression is de-repressed to substitute for defective adult hemoglobin in inherited diseases including beta-thalassemia and sickle-cell anemia. BCL11A has six CCHH-type zinc-fingers of which domains 4-6 are necessary and sufficient for dsDNA binding. Here, we focus on the CCHC-type ZNF at the N-terminus of BCL11A (residues 46-72), Z0, thought to modulate oligomerization of the transcription repressor. Using NMR and CD spectroscopy, Z0 is shown to be a thermostable CCHC zinc-finger with a pM dissociation constant for zinc. The NMR structure of Z0 has a prototypical beta-beta-alpha fold, with a hydrophobic knob comprising about half the structure. The unusual proportion of hydrophobic residues in Z0 led us to investigate if this is more general in zinc-fingers that do not bind dsDNA. We used the ZF and WebLogo servers to examine sequences of zinc fingers with demonstrated DNA-binding function, non-binders, and the CCHC-type family of protein-binders. DNA-binders are distinguished by contiguous stretches of high-scoring zinc-fingers. Non-DNA-binders show a depletion of polar residues at the positions expected to contact nucleotides and increased divergence from sequence consensus making these domains more likely to be annotated as atypical, degenerate, or to be missed as zinc-fingers. We anticipate these sequence patterns will help distinguish DNA-binders from non-binders, an open problem in the functional understanding of zinc-finger motifs.

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

Cited by 3 publications

References 74 publications

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Solution structure of the Z0 domain from transcription repressor BCL11A sheds light on the sequence properties of protein-binding zinc-fingers

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