Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

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Structural insights into single-stranded DNA binding and cleavage by F factor TraI

Conjugative plasmid transfer between bacteria disseminates antibiotic resistance and diversifies prokaryotic genomes. Relaxases, proteins essential for conjugation, cleave one plasmid strand sequence specifically prior to transfer. Cleavage occurs through a Mg(2+)-dependent transesterification involving a tyrosyl hydroxyl and a DNA phosphate. The structure of the F plasmid TraI relaxase domain, described here, is a five-strand beta sheet flanked by alpha helices. The protein resembles replication initiator protein AAV-5 Rep but is circularly permuted, yielding a different topology. The beta sheet forms a binding cleft lined with neutral, nonaromatic residues, unlike most single-stranded DNA binding proteins which use aromatic and charged residues. The cleft contains depressions, suggesting base recognition occurs in a knob-into-hole fashion. Unlike most nucleases, three histidines but no acidic residues coordinate a Mg(2+) located near the catalytic tyrosine. The full positive charge on the Mg(2+) and the architecture of the active site suggest multiple roles for Mg(2+) in DNA cleavage.     

Structural insights into RNA recognition by RIG-I

Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.     

Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23

Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.     

Structural insights into the PIP2 recognition by syntenin-1 PDZ domain

Lipid-mediated regulatory mechanism of the C-terminal ligand binding to PDZ domains is not fully understood, despite their roles in subcellular organization. Here, we provide structural insights into the phosphatidylinositol 4,5-bisphosphate (PIP₂) recognition mode of a PDZ domain, as revealed from the crystal structure of the phosphate-bound PDZ domain. Two adjacent phosphate ions bind to the basic residues close to the amino terminus of the α2 helix in the Tamalin PDZ domain, reflecting an interaction mode of the two phosphate groups of PIP₂. Based on the observed location of the two phosphate molecules within the PDZ domain, we built the docking model of PIP₂ with the PDZ domain of the well-known PIP₂-binding protein, syntenin-1. This model suggests that the hydrophobic diacylglycerol group of PIP₂ could contact the ligand-binding groove of the PDZ domain. These structural features well explain biological phenomena, which were previously reported for the PIP₂-mediated PDZ ligand-binding regulation.     

Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1

CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.     

Target DNA recognition and cleavage by a reconstituted Type I-G CRISPR-Cas immune effector complex

Extremophiles - CRISPR-Cas immune systems defend prokaryotes against viruses and plasmids. CRISPR RNAs (crRNAs) associate with various CRISPR-associated (Cas) protein modules to form structurally...     

Structural insights into lesion recognition and repair by the bacterial 8-oxoguanine DNA glycosylase MutM

MutM is a bacterial 8-oxoguanine glycosylase responsible for initiating base-excision repair of oxidized guanine residues in DNA. Here we report five different crystal structures of MutM-DNA complexes that represent different steps of the repair reaction cascade catalyzed by the protein and also differ in the identity of the base opposite the lesion (the 'estranged' base). These structures reveal that the MutM active site performs the multiple steps of base-excision and 3' and 5' nicking with minimal rearrangement of the DNA backbone.     

Structural insights into the recognition of substrates and activators by the OSR1 kinase

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signalling cascade regulating the activity of Na(+)/K(+)/2Cl(-) co-transporters (NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal (CCT) domain, which recognizes a unique peptide (Arg-Phe-Xaa-Val) motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as its substrates (NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-Phe-Xaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.     

Structural insights into recognition of acetylated histone ligands by the BRPF1 bromodomain

Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ HAT complex and contains a unique combination of domains typically found in chromatin-associated factors, which include plant homeodomain (PHD) fingers, a bromodomain and a proline-tryptophan-tryptophan-proline (PWWP) domain. Bromodomains are conserved structural motifs generally known to recognize acetylated histones, and the BRPF1 bromodomain preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We solved the X-ray crystal structures of the BRPF1 bromodomain in complex with the H2AK5ac and H4K12ac histone peptides. Site-directed mutagenesis on residues in the BRPF1 bromodomain-binding pocket was carried out to investigate the contribution of specific amino acids on ligand binding. Our results provide critical insights into the molecular mechanism of ligand binding by the BRPF1 bromodomain, and reveal that ordered water molecules are an essential component driving ligand recognition.     

Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX

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Crystallographic insights into DNA minor groove recognition by drugs

This review surveys the crystal structures between minor groove drugs and oligonucleotides, of which over thirty have now been determined. The various factors that are involved in the observed A/T sequence selectivity of these drugs are examined in structural terms. The roles of, in particular, hydrogen-bond recognition and sequence-dependent groove width, are assessed, and as a consequence the minor groove drugs have been classified into two categories, dependent on the relative roles played by these two factors in sequence recognition. Implications for the recognition of non-A/T sequences are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 44: 105–121, 1997     

Structural insights into the recognition of peroxisomal targeting signal 1 by Trypanosoma brucei peroxin 5

Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called “peroxins,” which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to have a residue in the α_L conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.     

Structural insights into DNA lesion bypass

A number of highly specialized DNA polymerases with the ability to replicate through DNA lesions have been identified. In this issue of Structure, Nair et al. show how one such polymerase, yeast Rev1, accomplishes the DNA lesion bypass task.     

Structural insights into the cofactor-assisted substrate recognition of yeast quinone oxidoreductase Zta1

Quinone oxidoreductase (QOR EC1.6.5.5) catalyzes the reduction of quinone to hydroxyquinone using NADPH as a cofactor. Here we present the crystal structure of the ζ-crystallin-like QOR Zta1 from Saccharomycescerevisiae in apo-form at 2.00 Å and complexed with NADPH at 1.59 Å resolution. Zta1 forms a homodimer, with each subunit containing a catalytic and a cofactor-binding domain. Upon NADPH binding to the interdomain cleft, the two domains shift towards each other, producing a better fit for NADPH, and tightening substrate binding. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis defined a potential quinone-binding site that determines the stringent substrate specificity. Moreover, multiple-sequence alignment and kinetics assays implied that a single-residue change from Arg in lower organisms to Gly in vertebrates possibly resulted in elevation of enzymatic activity of ζ-crystallin-like QORs throughout evolution.     

Structural insights into the regulation and the recognition of histone marks by the SET domain of NSD1

The development of epigenetic therapies fuels cancer hope. DNA-methylation inhibitors, histone-deacetylase and histone-methyltransferase (HMTase) inhibitors are being developed as the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. A growing number of studies have reported alterations or amplifications of NSD1, NSD2, or NSD3 in numerous carcinogenic events. Reducing NSDs activity through specific lysine-HMTase inhibitors appears promising to help suppressing cancer growth. However, little is known about the NSD pathways and our understanding of the histone lysine-HMTase mechanism is partial. To shed some light on both the recognition and the regulation of epigenetic marks by the SET domain of the NSD family, we investigate the structural mechanisms of the docking of the histone-H4 tail on the SET domain of NSD1. Our finding exposes a key regulatory and recognition mechanism driven by the flexibility of a loop at the interface of the SET and postSET region. Finally, we prospect the special value of this regulatory region for developing specific and selective NSD inhibitors for the epigenetic therapy of cancers.     

Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.