Biology subjects

Whitehead, R. D.

Publications and source records attributed to Whitehead, R. D..

5 recordsLinked to original sources

bioRxiv2024

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

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.

biophysics↗

bioRxiv2024

Structural Model of Bacteriophage P22 Scaffolding Protein in a Procapsid by Magic-Angle Spinning NMR

Icosahedral dsDNA viruses such as the tailed bacteriophages and herpesviruses have a conserved pathway to virion assembly that is initiated from a scaffolding protein driven procapsid formation. The dsDNA is actively packaged into procapsids, which undergo complex maturation reactions to form infectious virions. In bacteriophage P22, scaffolding protein (SP) directs the assembly of coat proteins into procapsids that have a T=7 icosahedral arrangement, en route to the formation of the mature P22 capsid. Other than the C-terminal helix-turn-helix involved in interaction with coat protein, the structure of the P22 303 amino acid scaffolding protein within the procapsid is not understood. Here, we present a structural model of P22 scaffolding protein encapsulated within the 23 MDa procapsid determined by magic angle spinning NMR spectroscopy. We took advantage of the 10-fold sensitivity gains afforded by the novel CPMAS CryoProbe to establish the secondary structure of P22 scaffolding protein and employed 19F MAS NMR experiments to probe its oligomeric state in the procapsid. Our results indicate that the scaffolding protein has both -helical and disordered segments and forms a trimer of dimers when bound to the procapsid lattice. This work provides the first structural information for P22 SP beyond the C-terminal helix-turn-helix and demonstrates the power of MAS NMR to understand higher-order viral protein assemblies involving structural components that are inaccessible to other structural biology techniques.

biochemistry↗

bioRxiv2023

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 {beta}{beta} tertiary structure with a pM dissociation constant for Zn2+, and a thermal stability >80 {degrees}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 domains DNA-binding properties and mutations associated with cancers. We expect other ZNFs currently classified as degenerate, are CCHC-type structures like Z*.

molecular biology↗

bioRxiv2022

Identification of a second site for coat protein binding in bacteriophage P22 scaffolding protein

Scaffolding proteins are essential for the assembly of most tailed, double-stranded DNA bacteriophages as well as herpesviruses. These proteins interact specifically with the coat proteins to efficiently assemble procapsids with the correct morphology. A helix-turn-helix (HTH) domain of bacteriophage P22 scaffolding protein is essential for coat binding, but the presence of additional coat protein binding sites has been predicted. An alanine substitution at scaffolding protein residue L245 causes a strong cold-sensitive phenotype. Both in vivo and in vitro assembly with L245A scaffolding protein yields aberrant and petite particles at non-permissive temperatures. The L245A scaffolding protein is destabilized as determined by thermal melts monitored by circular dichroism. Through crosslinking studies, residue L245 interacts with the coat protein A-domain residue D198, which has been predicted previously to contain a scaffolding protein binding site. L245 also binds R101 in the coat protein P-domain as well as E18 in the N-arm. These results demonstrate the presence of secondary coat binding sites that may function in conjunction with the HTH domain to promote the assembly of procapsids with the correct curvature. ImportanceMany dsDNA viruses, including tailed bacteriophages and Herpesviruses, assemble precursor capsids, or procapsids, using an essential catalytic scaffolding protein. How scaffolding proteins induce proper assembly of their major capsid proteins remains unclear. The scaffolding protein of bacteriophage P22 has a C-terminal helix-turn-helix domain that interacts with the N-arm of the coat protein to activate it for assembly. Here, a second potential coat protein interaction site is identified in scaffolding protein at residue L245. Residue L245 may be involved in stabilizing a small helical domain based the effect of the substitution scaffolding protein stability and a predicted model of the scaffolding protein fold, thereby indicating additional complexity in the interaction between coat and scaffolding proteins.

biochemistry↗

bioRxiv2019

NMR Mapping of Disordered Segments from a Viral Scaffolding Protein Encapsulated in a 23 MDa Procapsid Complex

Scaffolding proteins are requisite for the capsid shell assembly of many tailed dsDNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, no high-resolution structural information is available for scaffolding proteins within capsids. Here we use the inherent size limit of NMR to identify mobile segments of the phage P22 scaffolding protein in solution and when incorporated into a ~23 MDa procapsid complex. Free scaffolding protein gives NMR signals from both the N and C-terminus. When scaffolding protein is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix (HTH) domain disappear due to binding to the procapsid interior. Signals from the N-terminal domain persist, indicating 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 the dissociation and release of scaffolding protein, during the genome packaging step accompanying phage maturation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/539965v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@15a637aorg.highwire.dtl.DTLVardef@138e675org.highwire.dtl.DTLVardef@a24879org.highwire.dtl.DTLVardef@441cfb_HPS_FORMAT_FIGEXP M_FIG C_FIG Scaffolding protein (SP) nucleates the assembly of phage P22 coat proteins into an icosahedral capsid structure that envelops the viral genome. NMR spectra of free SP show signals from the N-terminus (red) and a helix-turn-helix domain at the C-terminus (blue). When SP is incorporated into empty phage P22 procapsids to form a 23 MDa complex, the subset of signals from the N-terminal 40 residues persist indicating this segment is disordered. The unfolded nature of the N-terminus coupled with its negatively charged character, is important for the functional requirement of SP to exit the capsid as it becomes packaged with its genome.

biophysics↗