Watching a signaling protein function: What has been learned over four decades of time-resolved studies of photoactive yellow protein.

Photoactive yellow protein (PYP) is a signaling protein whose internal p-coumaric acid chromophore undergoes reversible, light-induced trans-to-cis isomerization, which triggers a sequence of structural changes that ultimately lead to a signaling state. Since its discovery nearly 40 years ago, PYP has attracted much interest and has become one of the most extensively studied proteins found in nature. The method of time-resolved crystallography, pioneered by Keith Moffat, has successfully characterized intermediates in the PYP photocycle at near atomic resolution over 12 decades of time down to the sub-picosecond time scale, allowing one to stitch together a movie and literally watch a protein as it functions. But how close to reality is this movie? To address this question, results from numerous complementary time-resolved techniques including x-ray crystallography, x-ray scattering, and spectroscopy are discussed. Emerging from spectroscopic studies is a general consensus that three time constants are required to model the excited state relaxation, with a highly strained ground-state cis intermediate formed in less than 2.4 ps. Persistent strain drives the sequence of structural transitions that ultimately produce the signaling state. Crystal packing forces produce a restoring force that slows somewhat the rates of interconversion between the intermediates. Moreover, the solvent composition surrounding PYP can influence the number and structures of intermediates as well as the rates at which they interconvert. When chloride is present, the PYP photocycle in a crystal closely tracks that in solution, which suggests the epic movie of the PYP photocycle is indeed based in reality.

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C -terminal domains (Rpn S ), which are translated separately from the full-length proteins (Rpn L ), directly block the activities of the toxic full-length proteins. The crystal structure of RpnA S revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2 L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. Significance: Here we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longer rpn genes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.

Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding.

The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end.

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells.

DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5'-transposon end.

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.

Structural Basis for a Dual Function ATP Grasp Ligase That Installs Single and Bicyclic ω-Ester Macrocycles in a New Multicore RiPP Natural Product.

Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, "graspetides" (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a "hairpin topology" with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.

Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA transposition.

Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.

Structural basis of seamless excision and specific targeting by piggyBac transposase.

The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.

Comment on "RNA-guided DNA insertion with CRISPR-associated transposases".

Strecker et al (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.

Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.

Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase.

Some DNA transposons relocate from one genomic location to another using a mechanism that involves generating double-strand breaks at their transposon ends by forming hairpins on flanking DNA. The same double-strand break mode is employed by the V(D)J recombinase at signal-end/coding-end junctions during the generation of antibody diversity. How flanking hairpins are formed during DNA transposition has remained elusive. Here, we describe several co-crystal structures of the Hermes transposase bound to DNA that mimics the reaction step immediately prior to hairpin formation. Our results reveal a large DNA conformational change between the initial cleavage step and subsequent hairpin formation that changes which strand is acted upon by a single active site. We observed that two factors affect the conformational change: the complement of divalent metal ions bound by the catalytically essential DDE residues, and the identity of the -2 flanking base pair. Our data also provides a mechanistic link between the efficiency of hairpin formation (an A:T basepair is favored at the -2 position) and Hermes' strong target site preference. Furthermore, we have established that the histidine residue within a conserved C/DxxH motif present in many transposase families interacts directly with the scissile phosphate, suggesting a crucial role in catalysis.

Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering.

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction network between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions.

A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity.

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition.

Helitrons are eukaryotic DNA transposons that have profoundly affected genome variability via capture and mobilization of host genomic sequences. Defining their mode of action is therefore important for understanding how genome landscapes evolve. Sequence similarities with certain prokaryotic mobile elements suggest a "rolling circle" mode of transposition, involving only a single transposon strand. Using the reconstituted Helraiser transposon to study Helitron transposition in cells and in vitro, we show that the donor site must be double-stranded and that single-stranded donors will not suffice. Nevertheless, replication and integration assays demonstrate the use of only one of the transposon donor strands. Furthermore, repeated reuse of Helraiser donor sites occurs following DNA synthesis. In cells, circular double-stranded intermediates that serve as transposon donors are generated and replicated by Helraiser transposase. Cell-free experiments demonstrate strand-specific cleavage and strand transfer, supporting observations made in cells.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids.

The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. IMPORTANCE: The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as transposons and integrons have been strongly associated with the rapid spread of genes responsible for antibiotic resistance. Understanding the consequences of their actions allowed us to establish unambiguous evolutionary relationships between plasmids in the analysis set.