Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling.

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.

Differential contributions of the latch in Thermotoga maritima reverse gyrase to the binding of single-stranded DNA before and after ATP hydrolysis.

Reverse gyrase catalyzes the ATP-dependent introduction of positive supercoils into DNA. Supercoiling requires the functional cooperation of its N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain contains a superfamily 2 helicase core formed by two RecA domains, H1 and H2. We show here that a helicase domain lacking the latch, an insertion in H2, fails to close the cleft in the helicase core in response to nucleotide and DNA binding at the beginning of the catalytic cycle. In the presence of the pre-hydrolysis ATP analog ADP·BeFx, however, the closed conformer can still be formed in the absence of the latch. The helicase domain lacking the latch exhibits reduced DNA affinities. The energetic difference between the two nucleotide states involved in duplex separation is diminished, rationalizing the unwinding deficiency of reverse gyrase lacking the latch. The latch most strongly contributes to binding of single-stranded DNA in the post-hydrolysis state, before phosphate release. Our results are in line with contributions of the latch in determining the direction of strand passage, and in orienting the cleaved single-stranded DNA for re-ligation. At the same time, the latch may coordinate the re-ligation reaction with strand passage and with the nucleotide cycle.

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling.

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain.

The conformational flexibility of the helicase-like domain from Thermotoga maritima reverse gyrase is restricted by the topoisomerase domain.

Reverse gyrase is the only enzyme known to introduce positive supercoils into DNA. Positive supercoiling is achieved by the functional cooperation of a helicase-like and a topoisomerase domain. The isolated helicase-like domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. In the context of reverse gyrase, these individual activities are suppressed or attenuated. The helicase-like domain of Thermotoga maritima reverse gyrase is a nucleotide-dependent conformational switch that binds DNA and ATP cooperatively. It provides a nucleotide-dependent DNA-binding site to reverse gyrase and thus serves as a valuable model for the investigation of the effect of nucleotides on DNA processing by reverse gyrase that is key to its supercoiling activity. To improve our understanding of the structural basis for the functional cooperation of a helicase domain with a DNA topoisomerase, we have determined the structures of the isolated helicase-like domain of T. maritima reverse gyrase in five different conformations. Comparison of these structures reveals extensive domain flexibility in the absence of conformational restrictions by the topoisomerase that is consistent with single-molecule Förster resonance energy transfer experiments presented here. The structure of the first ADP-bound form provides novel details about nucleotide binding to reverse gyrase. It demonstrates that reverse gyrases use the canonical nucleotide binding mode common to superfamily 2 helicases despite large deviations in the conserved motifs. A characteristic insert region adopts drastically different structures in different reverse gyrases. Counterparts of this insert region are located at very different positions in other DNA-processing enzymes but may point toward a general role in DNA strand separation.

Nucleotide-driven conformational changes in the reverse gyrase helicase-like domain couple the nucleotide cycle to DNA processing.

Reverse gyrase introduces positive supercoils into DNA in an ATP-dependent process. It has a modular structure comprising a helicase-like and a topoisomerase domain. The helicase-like domain consists of two RecA-like subdomains and thus structurally resembles members of the helicase superfamily 2. It is a nucleotide-dependent switch that alters between an ATP state with a slight preference for dsDNA, and an ADP state with a high preference for ssDNA. Inter-domain communication between the helicase-like and the topoisomerase domain is central for their functional cooperation in reverse gyrase. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the nucleotide cycle of the reverse gyrase helicase-like domain in the absence and presence of different DNA substrates. With this comprehensive thermodynamic characterization of the nucleotide cycle of the helicase-like domain, in combination with single molecule FRET data on the conformation of the helicase-like domain at all stages of the catalytic cycle, a picture emerges as to how the helicase-like domain may guide ATP-dependent positive supercoiling by reverse gyrase.

The reverse gyrase helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain.

Reverse gyrase is a topoisomerase that introduces positive supercoils into DNA in an ATP-dependent manner. It is unique to hyperthermophilic archaea and eubacteria, and has been proposed to protect their DNA from damage at high temperatures. Cooperation between its N-terminal helicase-like and the C-terminal topoisomerase domain is required for positive supercoiling, but the precise role of the helicase-like domain is currently unknown. Here, the characterization of the isolated helicase-like domain from Thermotoga maritima reverse gyrase is presented. We show that the helicase-like domain contains all determinants for nucleotide binding and ATP hydrolysis. Its intrinsic ATP hydrolysis is significantly stimulated by ssDNA, dsDNA and plasmid DNA. During the nucleotide cycle, the helicase-like domain switches between high- and low-affinity states for dsDNA, while its affinity for ssDNA in the ATP and ADP states is similar. In the context of reverse gyrase, the differences in DNA affinities of the nucleotide states are smaller, and the DNA-stimulated ATPase activity is strongly reduced. This inhibitory effect of the topoisomerase domain decelerates the progression of reverse gyrase through the nucleotide cycle, possibly providing optimal coordination of ATP hydrolysis with the complex reaction of DNA supercoiling.

MDA5-filament, dynamics and disease.

Melanoma Differentiation-Associated gene 5 (MDA5), encoded by the gene IFIH1, is a cytoplasmic sensor for viral double-stranded RNAs (dsRNAs). MDA5 activates the type I interferon signaling pathway upon detection of long viral dsRNA generated during replication of picornaviruses. Studies have shown that MDA5 forms a filament along the length of dsRNA and utilizes ATP-dependent filament dynamics to discriminate between self versus non-self on the basis of dsRNA length. This review summarizes our current understanding of how the MDA5 filament assembles and disassembles, how this filament dynamics are utilized in dsRNA length-dependent signaling, and how dysregulated filament dynamics lead to pathogenesis of immune disorders.

Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I.

UNLABELLED: Stimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5'-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG(70-114)) that is essential for the potent immunostimulatory activity of 5'-triphosphate-containing SeV iDVGs. DVG(70-114) enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG(70-114) augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA. IMPORTANCE: A discrete group of molecular motifs, including 5'-triphosphates associated with double-stranded RNA, have been identified as essential for the triggering of antiviral immunity. Most RNA viruses expose these motifs during their replication; however, successful viruses normally evade immune recognition and replicate to high levels before detection, indicating that unknown factors drive antiviral immunity. DVGs from SeV are among the most potent natural viral stimuli of the antiviral response known to date. These studies define a new natural viral motif present in DVGs that maximizes viral recognition by the intracellular sensor RIG-I, allowing fast and strong antiviral responses even in the presence of viral-encoded immune antagonists. This motif can be harnessed to increase the immunostimulatory potential of otherwise inert viral RNAs and represents a novel immunostimulatory enhancer that could be used in the development of vaccine adjuvants and antivirals.

Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling.

The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome and of other undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (also called MDA5) cause a spectrum of neuroimmunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer gain of function such that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.

Reverse gyrase transiently unwinds double-stranded DNA in an ATP-dependent reaction.

Reverse gyrase is a unique DNA topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. It consists of a helicase domain that functionally cooperates with a topoisomerase domain. Different models for the catalytic mechanism of reverse gyrase that predict a central role of the helicase domain have been put forward. The helicase domain acts as a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. It has been suggested that the helicase domain can unwind double-stranded regions, but helicase activity has not been demonstrated as yet. Here, we show that the isolated helicase domain and full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain, is required for DNA supercoiling. Strikingly, the helicase domain lacking the latch cannot unwind DNA, linking unwinding to DNA supercoiling. The unwinding activity may provide and stabilize the single-stranded regions required for strand passage by the topoisomerase domain, either de novo or by expanding already existing unpaired regions that may form at high temperatures.