CTCF, a conserved 3D genome architecture protein, determines proper genome-wide chromatin looping interactions through directional binding to specific sequence elements of four modules within ...numerous CTCF-binding sites （CBSs） by its 11 zinc fingers （ZFs）. Here, we report four crystal structures of human CTCF in complex with CBSs of the protocadherin （Pcdh） clusters. We show that directional CTCF binding to cognate CBSs of the Pcdh enhancers and promoters is achieved through inserting its ZF3, ZFs 4-7, and ZFs 9-11 into the major groove along CBSs, result- ing in a sequence-specific recognition of module 4, modules 3 and 2, and module 1, respectively; and ZF8 serves as a spacer element for variable distances between modules 1 and 2. In addition, the base contact with the asymmetric ＂A＂ in the central position of modules 2-3, is essential for directional recognition of the CBSs with symmetric core sequences but lacking module 1. Furthermore, CTCF tolerates base changes at specific positions within the degen- erated CBS sequences, permitting genome-wide CTCF binding to a diverse range of CBSs. Together, these complex structures provide important insights into the molecular mechanisms for the directionality, diversity, flexibility, dynamics, and conservation of multivalent CTCF binding to its cognate sites across the entire human genome.
CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems provide prokaryotic cells with adaptive immunity against invading bacteriophages. ...Bacteriophages counteract bacterial responses by encoding anti-CRISPR inhibitor proteins (Acr). However, the structural basis for their inhibitory actions remains largely unknown. Here, we report the crystal structure of the AcrIIA2-SpyCas9-sgRNA (single-guide RNA) complex at 3.3 Å resolution. We show that AcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region. More specifically, AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection. Thus, phage-encoded AcrIIA2 appears to act as a DNA mimic that blocks subsequent dsDNA binding by virtue of its highly acidic residues, disabling bacterial Cas9 by competing with target dsDNA binding with a binding motif distinct from AcrIIA4. Our study provides a more detailed mechanistic understanding of AcrIIA2-mediated inhibition of SpyCas9, the most widely used genome-editing tool, opening new avenues for improved regulatory precision during genome editing.
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•Crystal structure of SpyCas9 in complex with sgRNA and suppressor AcrIIA2•AcrIIA2 recognizes sgRNA-bound SpyCas9•AcrIIA2 prevents the target DNA search by blocking the PAM recognition residues•AcrIIA2 disables bacterial Cas9 by competing with target dsDNA binding
Liu et al. determined the crystal structure of the AcrIIA2-SpyCas9-sgRNA complex. The structure reveals that AcrIIA2 acts as a DNA mimic blocking subsequent dsDNA recognition and binding.
Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of ...Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3′ overhang segments. The PAM-complementary sequence in the 3′ overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.
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•The dual-forked protospacer is integrated via a cut-and-paste mechanism•Architecture of Cas1-Cas2 predetermines length of newly acquired spacer•Cas1a recognizes PAM-complementary sequence via sequence-specific interactions•Cas1-Cas2 undergoes a conformational change upon protospacer DNA binding
Cas1 and Cas2 select an invading DNA sequence, termed protospacer, for insertion into the CRISPR locus of the host cell. The structure of the Cas1-Cas2-protospacer DNA complex reveals the dual-forked nature of the protospacer, explains how the protospacer is selected, and identifies how protospacer length is predetermined.
C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide ...RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.
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•Crystal structure of Alicyclobacillus acidoterrestris C2c1-sgRNA complex•Mechanistic insights into dual-RNA-guided, C2c1-catalyzed, staggered dsDNA breaks•High mismatch sensitivity of C2c1 reduces off-target cleavage•Join 2/4-truncated sgRNA-guided DNA cleavage similar to that of wild-type sgRNA
Liu et al. report the structure of C2c1-a type V-B CRISPR-Cas endonuclease, in complex with a chimeric single guide RNA, providing insights into the high specificity of DNA cleavage by C2c1. C2c1’s low off-target rate makes it a valuable addition to the current arsenal of gene-editing tools.
C2c2, the effector of type VI CRISPR-Cas systems, has two RNase activities—one for cutting its RNA target and the other for processing the CRISPR RNA (crRNA). Here, we report the structures of ...Leptotrichia shahii C2c2 in its crRNA-free and crRNA-bound states. While C2c2 has a bilobed structure reminiscent of all other Class 2 effectors, it also exhibits different structural characteristics. It contains the REC lobe with a Helical-1 domain and the NUC lobe with two HEPN domains. The two RNase catalytic pockets responsible for cleaving pre-crRNA and target RNA are independently located on Helical-1 and HEPN domains, respectively. crRNA binding induces significant conformational changes that are likely to stabilize crRNA binding and facilitate target RNA recognition. These structures provide important insights into the molecular mechanism of dual RNase activities of C2c2 and establish a framework for its future engineering as a RNA editing tool.
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•Crystal structure of Leptotrichia shahii C2c2-crRNA binary complex and apo form•A bulge-containing crRNA stem is essential for C2c2 RNase activities•Helical-1 domain has the catalytic site for pre-crRNA processing•Promiscuous RNA cleavage due to distance between HEPN catalytic site and crRNA guide
The structural analysis of C2c2 reveals that its dual RNase catalytic sites locate independently in two physically distant domains, explaining its promiscuous cleavage of target RNA and specific processing of pre-CRISPR RNA.
The zinc-finger (ZnF) repressor protein BCL11A regulates the switch from fetal (HbF, α 2γ 2) to adult hemoglobin (HbA, α 2β 2) through direct binding at the γ-globin promoter. Clinical trials of ...gene-based reduction of BCL11A with ex vivo modified hematopoietic stem cells (HSCs) have yielded transformative outcomes in patients with sickle cell disease and β-thalassemia, establishing BCL11A as a therapeutic target. Unless in vivo methods for delivery of gene-based therapies become efficient enough to modify a substantial fraction of host HSCs, small molecule drugs will be required to reduce overall disease burden. With the goal of targeting BCL11A with small molecules, we have characterized its native state. The protein is largely unstructured, except for 7 ZnFs, numbered 0-6. Previously we showed that BCL11A protein appears stable in cells with a T 1/2 of ~ 24 hrs. Here we report the unexpected finding that the protein assembles and functions as a tetramer. Recombinant BCL11A behaves as a tetramer in biophysical assays. Gene editing of the endogenous BCL11A locus in CD34-derived, immortal HUDEP2 cells has been employed to correlate protein structure and expression with consequences of a given modification. We found that BCL11A lacking ZnF0 is unstable, leading to protein loss and derepression of γ-globin. Recombinant ZnF0 self-assembles into a stable tetramer, whose X-ray structure reveals a hydrophobic interface between subunits. Mutation of residues in the hydrophobic core disrupts tetramerization and markedly reduces steady-state protein in cells. Reciprocal immunoprecipitation in cells reveals that ZnF0 is both necessary and sufficient to mediate BCL11A mutlimer assembly. Collectively, these results indicate that BCL11A tetramerization is required for protein stability. In cells expressing BCL11A lacking ZnF0, the steady-state protein level is partially restored by addition of proteasome inhibitors, suggesting that mutant protein is actively degraded by the ubiquitin-proteasome system. In contrast to findings with ZnF0, discrete removal of ZnF1 within the context of full-length BCL11A results in a marked increase in steady-state protein in cells. In vitro purified ZnF1 forms a dimer and only dimeric ZnF1 can be stably expressed in cells. These findings re consistent with a model in which assembly of the BCL11A tetramer is required to mask a degron in ZnF1 that becomes obscured on its dimerization. Masking of a degron in ZnF1 predicts that discrete removal of both ZnF0 and ZnF1 within full-length BCL11A will lead to production of a monomer. Indeed, HUDEP2 cells expressing BCL11A lacking both ZnFs exhibit an elevated level of steady-state, monomeric protein, consistent with the degron masking model. However, despite an elevated level, cells expressing monomeric BCL11A are defective in HbF silencing, indicating that the tetramer structure is critical for γ-globin repression. Our findings provide novel insights into BCL11A action and suggest new strategies for therapeutic targeting. Combining structural, biochemical, and cellular approaches, we have shown that ZnF-mediated tetramer formation shields BCL11A from proteolytic degradation, and that the tetramer state itself is required for effective γ-globin repression. Of relevance to BCL11A as a therapeutic target, the tetramer of ZnF0 constitutes a structured, stable domain in an otherwise largely unstructured protein, and thereby offers possibilities for small molecule targeting. The multimer state of BCL11A is a vulnerability for HbF repression.
Evolution of nanobodies specific for BCL11A Yin, Maolu; Izadi, Manizheh; Tenglin, Karin ...
Proceedings of the National Academy of Sciences - PNAS,
01/2023, Letnik:
120, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to ...the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Here, we describe the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. We obtained first-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) from a synthetic yeast surface display library, and employed error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA-binding specificity. Given the ease of manipulation of nanobodies and their exquisite specificity, nanobody-mediated TPD of TFs should be suitable for dissecting regulatory relationships of TFs and gene targets and validating therapeutic potential of proteins of interest.
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