Structural and functional characterization of cyclic pyrimidine-regulated anti-phage system.

3',5'-cyclic uridine monophosphate (cUMP) and 3',5'-cyclic cytidine monophosphate (cCMP) have been established as bacterial second messengers in the phage defense system, named pyrimidine cyclase system for anti-phage resistance (Pycsar). This system consists of a pyrimidine cyclase and a cyclic pyrimidine receptor protein. However, the molecular mechanism underlying cyclic pyrimidine synthesis and recognition remains unclear. Herein, we determine the crystal structures of a uridylate cyclase and a cytidylate cyclase, revealing the conserved residues for cUMP and cCMP production, respectively. In addition, a distinct zinc-finger motif of the uridylate cyclase is identified to confer substantial resistance against phage infections. Furthermore, structural characterization of cUMP receptor protein PycTIR provides clear picture of specific cUMP recognition and identifies a conserved N-terminal extension that mediates PycTIR oligomerization and activation. Overall, our results contribute to the understanding of cyclic pyrimidine-mediated bacterial defense.

Structural insights into the regulation, ligand recognition, and oligomerization of bacterial STING.

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling pathway plays a critical protective role against viral infections. Metazoan STING undergoes multilayers of regulation to ensure specific signal transduction. However, the mechanisms underlying the regulation of bacterial STING remain unclear. In this study, we determined the crystal structure of anti-parallel dimeric form of bacterial STING, which keeps itself in an inactive state by preventing cyclic dinucleotides access. Conformational transition between inactive and active states of bacterial STINGs provides an on-off switch for downstream signaling. Some bacterial STINGs living in extreme environment contain an insertion sequence, which we show codes for an additional long lid that covers the ligand-binding pocket. This lid helps regulate anti-phage activities. Furthermore, bacterial STING can bind cyclic di-AMP in a triangle-shaped conformation via a more compact ligand-binding pocket, forming spiral-shaped protofibrils and higher-order fibril filaments. Based on the differences between cyclic-dinucleotide recognition, oligomerization, and downstream activation of different bacterial STINGs, we proposed a model to explain structure-function evolution of bacterial STINGs.

Crystal structure and functional implications of cyclic di-pyrimidine-synthesizing cGAS/DncV-like nucleotidyltransferases.

Purine-containing nucleotide second messengers regulate diverse cellular activities. Cyclic di-pyrimidines mediate anti-phage functions in bacteria; however, the synthesis mechanism remains elusive. Here, we determine the high-resolution structures of cyclic di-pyrimidine-synthesizing cGAS/DncV-like nucleotidyltransferases (CD-NTases) in clade E (CdnE) in its apo, substrate-, and intermediate-bound states. A conserved (R/Q)xW motif controlling the pyrimidine specificity of donor nucleotide is identified. Mutation of Trp or Arg from the (R/Q)xW motif to Ala rewires its specificity to purine nucleotides, producing mixed purine-pyrimidine cyclic dinucleotides (CDNs). Preferential binding of uracil over cytosine bases explains the product specificity of cyclic di-pyrimidine-synthesizing CdnE to cyclic di-UMP (cUU). Based on the intermediate-bound structures, a synthetic pathway for cUU containing a unique 2'3'-phosphodiester linkage through intermediate pppU[3'-5']pU is deduced. Our results provide a framework for pyrimidine selection and establish the importance of conserved residues at the C-terminal loop for the specificity determination of CD-NTases.

Specific recognition of cyclic oligonucleotides by Cap4 for phage infection.

Under selective pressure, bacteria have evolved diverse defense systems against phage infections. The SMODS-associated and fused to various effector domains (SAVED)-domain containing proteins were identified as major downstream effectors in cyclic oligonucleotide-based antiphage signaling system (CBASS) for bacterial defense. Recent study structurally characterizes a cGAS/DncV-like nucleotidyltransferase (CD-NTase)-associated protein 4 from Acinetobacter baumannii (AbCap4) in complex with 2'3'3'-cyclic AMP-AMP-AMP (cAAA). However, the homologue Cap4 from Enterobacter cloacae (EcCap4) is activated by 3'3'3'-cyclic AMP-AMP-GMP (cAAG). To elucidate the ligand specificity of Cap4 proteins, we determined the crystal structures of full-length wild-type and K74A mutant of EcCap4 to 2.18 and 2.42 Å resolution, respectively. The DNA endonuclease domain of EcCap4 shares similar catalytic mechanism with type II restriction endonuclease. Mutating the key residue K74 in the conserved DXn(D/E)XK motif completely abolishes its DNA degradation activity. The potential ligand-binding cavity of EcCap4 SAVED domain is located adjacent to its N-terminal domain, significantly differing from the centrally located cavity of AbCap4 SAVED domain which recognizes cAAA. Based on structural and bioinformatic analysis, we found that Cap4 proteins can be classified into two types: the type I Cap4, like AbCap4, recognize cAAA and the type II Cap4, like EcCap4, bind cAAG. Several conserved residues identified at the surface of potential ligand-binding pocket of EcCap4 SAVED domain are confirmed by ITC experiment for their direct binding roles for cAAG. Changing Q351, T391 and R392 to alanine abolished the binding of cAAG by EcCap4 and significantly reduced the anti-phage ability of the E. cloacae CBASS system constituting EcCdnD (CD-NTase in clade D) and EcCap4. In summary, we revealed the molecular basis for specific cAAG recognition by the C-terminal SAVED domain of EcCap4 and demonstrates the structural differences for ligand discrimination among different SAVED-domain containing proteins.

Crystal structure of the capsular polysaccharide-synthesis enzyme CapG from Staphylococcus aureus.

Bacterial capsular polysaccharides provide protection against environmental stress and immune evasion from the host immune system, and are therefore considered to be attractive therapeutic targets for the development of anti-infectious reagents. Here, we focused on CapG, one of the key enzymes in the synthesis pathway of capsular polysaccharides type 5 (CP5) from the opportunistic pathogen Staphylococcus aureus. SaCapG catalyses the 2-epimerization of UDP-N-acetyl-D-talosamine (UDP-TalNAc) to UDP-N-acetyl-D-fucosamine (UDP-FucNAc), which is one of the nucleotide-activated precursors for the synthesis of the trisaccharide repeating units of CP5. Here, the cloning, expression and purification of recombinant SaCapG are reported. After extensive efforts, single crystals of SaCapG were successfully obtained which belonged to space group C2 and exhibited unit-cell parameters a = 302.91, b = 84.34, c = 145.09 Å, ß = 110.65°. The structure was solved by molecular replacement and was refined to 3.2 Šresolution. The asymmetric unit revealed a homohexameric assembly of SaCapG, which was consistent with gel-filtration analysis. Structural comparison with UDP-N-acetyl-D-glucosamine 2-epimerase from Methanocaldococcus jannaschii identified α2, the α2-α3 loop and α10 as a gate-regulated switch controlling substrate entry and/or product release.

Peimine inhibits variants of SARS-CoV-2 cell entry via blocking the interaction between viral spike protein and ACE2.

Coronavirus disease 2019 (COVID-19) is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several vaccines against SARS-CoV-2 have been approved; however, variants of concern (VOCs) can evade vaccine protection. Therefore, developing small compound drugs that directly block the interaction between the viral spike glycoprotein and ACE2 is urgently needed to provide a complementary or alternative treatment for COVID-19 patients. We developed a viral infection assay to screen a library of approximately 126 small molecules and showed that peimine inhibits VOCs viral infections. In addition, a fluorescence resonance energy transfer (FRET) assay showed that peimine suppresses the interaction of spike and ACE2. Molecular docking analysis revealed that peimine exhibits a higher binding affinity for variant spike proteins and is able to form hydrogen bonds with N501Y in the spike protein. These results suggest that peimine, a compound isolated from Fritillaria, may be a potent inhibitor of SARS-CoV-2 variant infection. PRACTICAL APPLICATIONS: In this study, we identified a naturally derived compound of peimine, a major bioactive alkaloid extracted from Fritillaria, that could inhibit SARS-CoV-2 variants of concern (VOCs) viral infection in 293T/ACE2 and Calu-3 lung cells. In addition, peimine blocks viral entry through interruption of spike and ACE2 interaction. Moreover, molecular docking analysis demonstrates that peimine has a higher binding affinity on N501Y in the spike protein. Furthermore, we found that Fritillaria significantly inhibits SARS-CoV-2 viral infection. These results suggested that peimine and Fritillaria could be a potential functional drug and food for COVID-19 patients.

Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

Crystal structure and functional implication of bacterial STING.

Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in ß-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.

Hesperidin Is a Potential Inhibitor against SARS-CoV-2 Infection.

Hesperidin (HD) is a common flavanone glycoside isolated from citrus fruits and possesses great potential for cardiovascular protection. Hesperetin (HT) is an aglycone metabolite of HD with high bioavailability. Through the docking simulation, HD and HT have shown their potential to bind to two cellular proteins: transmembrane serine protease 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2), which are required for the cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results further found that HT and HD suppressed the infection of VeroE6 cells using lentiviral-based pseudo-particles with wild types and variants of SARS-CoV-2 with spike (S) proteins, by blocking the interaction between the S protein and cellular receptor ACE2 and reducing ACE2 and TMPRSS2 expression. In summary, hesperidin is a potential TMPRSS2 inhibitor for the reduction of the SARS-CoV-2 infection.

Scutellaria barbata D. Don Inhibits the Main Proteases (Mpro and TMPRSS2) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic emerged to severely impact the global population, creating an unprecedented need for effective treatments. This study aims to investigate the potential of Scutellaria barbata D. Don (SB) as a treatment for SARS-CoV-2 infection through the inhibition of the proteases playing important functions in the infection by SARS-CoV-2. FRET assay was applied to investigate the inhibitory effects of SB on the two proteases involved in SARS-CoV-2 infection, Mpro and TMPRSS2. Additionally, to measure the potential effectiveness of SB treatment on infection inhibition, cellular models based on the Calu3 and VeroE6 cells and their TMPRSS2- expressing derivatives were assessed by viral pseudoparticles (Vpp) infection assays. The experimental approaches were conjugated with LC/MS analyses of the aqueous extracts of SB to identify the major constituent compounds, followed by a literature review to determine the potential active components of the inhibitory effects on protease activities. Our results showed that SB extracts inhibited the enzyme activities of Mpro and TMPRSS2. Furthermore, SB extracts effectively inhibited SARS-CoV-2 Vpp infection through a TMPRSS2-dependent mechanism. The aqueous extract analysis identified six major constituent compounds present in SB. Some of them have been known associated with inhibitory activities of TMPRSS2 or Mpro. Thus, SB may effectively prevent SARS-CoV-2 infection and replication through inhibiting Mpro and TMPRSS2 protease activities.

Screening strategy of TMPRSS2 inhibitors by FRET-based enzymatic activity for TMPRSS2-based cancer and COVID-19 treatment.

Transmembrane serine protease (TMPRSS2) plays an oncogenic role in prostate cancer as the fusion gene with ERG, and has also been demonstrated to be essential for the cellular entry of severe acute respiratory syndrome coronaviruses (SARS-CoV). Thus, targeting TMPRSS2 is a promising strategy for therapies against both prostate cancer and coronavirus infection. Although Nafamostat and Camostat have been identified as TMPRSS2 inhibitors, severe side effects such as cerebral hemorrhage, anaphylactoid reaction, and cardiac arrest shock greatly hamper their clinical use. Therefore, more potent and safer drugs against this serine protease should be further developed. In this study, we developed a fluorescence resonance energy transfer (FRET)-based platform for effectively screening of inhibitors against TMPRSS2 protease activity. The disruption of FRET between green and red fluorescent proteins conjugated with the substrate peptide, which corresponds to the cleavage site of SARS-CoV-2 Spike protein, was measured to determine the enzymatic activity of TMPRSS2. Through an initiate pilot screening with around 100 compounds, Flupirtine, a selective neuronal potassium channel opener, was identified as a potential TMPRSS2 inhibitor from an FDA-approved drug library by using this screening platform, and showed inhibitory effect on the TMPRSS-dependent infection of SARS-CoV-2 Spike-pseudotyped lentiviral particles. This study describes a platform proven effective for rapidly screening of TMPRSS2 inhibitors, and suggests that Flupirtine may be worthy of further consideration of repurposing to treat COVID-19 patients.

Crystal structure and functional implication of a bacterial cyclic AMP-AMP-GMP synthetase.

Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.

Crystal structure of the N-terminal domain of TagH reveals a potential drug targeting site.

Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting.

Structural basis of SARS-CoV-2 main protease inhibition by a broad-spectrum anti-coronaviral drug.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or 2019 novel coronavirus (2019-nCoV), took tens of thousands of lives and caused tremendous economic losses. The main protease (Mpro) of SARS-CoV-2 is a potential target for treatment of COVID-19 due to its critical role in maturation of viral proteins and subsequent viral replication. Conceptually and technically, targeting therapy against Mpro is similar to target therapy to treat cancer. Previous studies show that GC376, a broad-spectrum dipeptidyl Mpro inhibitor, efficiently blocks the proliferation of many animal and human coronaviruses including SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), porcine epidemic diarrhea virus (PEDV), and feline infectious peritonitis virus (FIPV). Due to the conservation of structure and catalytic mechanism of coronavirus main protease, repurposition of GC376 against SARS-CoV-2 may be an effective way for the treatment of COVID-19 in humans. To validate this conjecture, the binding affinity and IC50 value of Mpro with GC376 was determined by isothermal titration calorimetry (ITC) and fluorescence resonance energy transfer (FRET) assay, respectively. The results showed that GC376 binds to SARS-CoV-2 Mpro tightly (KD = 1.6 µM) and efficiently inhibit its proteolytic activity (IC50 = 0.89 µM). We also elucidate the high-resolution structure of dimeric SARS-CoV-2 Mpro in complex with GC376. The cocrystal structure showed that GC376 and the catalytic Cys145 of Mpro covalently linked through forming a hemithioacetal group and releasing a sulfonic acid group. Because GC376 is already known as a broad-spectrum antiviral medication and successfully used in animal, it will be a suitable candidate for anti-COVID-19 treatment.

Tannic acid suppresses SARS-CoV-2 as a dual inhibitor of the viral main protease and the cellular TMPRSS2 protease.

The cell surface protein TMPRSS2 (transmembrane protease serine 2) is an androgen-responsive serine protease important for prostate cancer progression and therefore an attractive therapeutic target. Besides its role in tumor biology, TMPRSS2 is also a key player in cellular entry by the SARS-CoV viruses. The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has resulted in huge losses in socio-economy, culture, and human lives for which safe and effective cures are highly demanded. The main protease (Mpro/3CLpro) of SARS-CoV-2 is a critical enzyme for viral propagation in host cells and, like TMPRSS2, has been exploited for treatment of the infectious disease. Numerous natural compounds abundant in common fruits have been suggested with anti-coronavirus infection in the previous outbreaks of SARS-CoV. Here we show that screening of these compounds identified tannic acid a potent inhibitor of both SARS-CoV-2 Mpro and TMPRSS2. Molecular analysis demonstrated that tannic acid formed a thermodynamically stable complex with the two proteins at a KD of 1.1 mM for Mpro and 1.77 mM for TMPRSS2. Tannic acid inhibited the activities of the two proteases with an IC50 of 13.4 mM for Mpro and 2.31 mM for TMPRSS2. Mpro protein. Consistently, functional assays using the virus particles pseudotyped (Vpp) of SARS-CoV2-S demonstrated that tannic acid suppressed viral entry into cells. Thus, our results demonstrate that tannic acid has high potential of developing anti-COVID-19 therapeutics as a potent dual inhibitor of two independent enzymes essential for SARS-CoV-2 infection.

The tetrameric structure of sialic acid-synthesizing UDP-GlcNAc 2-epimerase from Acinetobacter baumannii: A comparative study with human GNE.

Sialic acid presentation on the cell surface by some pathogenic strains of bacteria allows their escape from the host immune system. It is one of the major virulence factors. Bacterial biosynthesis of sialic acids starts with the conversion of UDP-GlcNAc to UDP and ManNAc by a hydrolyzing 2-epimerase. Here, we present the crystal structure of this enzyme, named NeuC, from Acinetobacter baumannii The protein folds into two Rossmann-like domains and forms dimers and tetramers as does the epimerase part of the bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). In contrast to human GNE, which showed only the closed conformation, the NeuC crystals contained both open and closed protomers in each dimer. Substrate soaking changed the space group from C2221 to P212121 In addition to UDP, an intermediate-like ligand was seen bound to the closed protomer. The UDP-binding mode in NeuC was similar to that in GNE, although a few side chains were rotated away. NeuC lacks the CMP-Neu5Ac-binding site for allosteric inhibition of GNE. However, the two enzymes as well as other NeuC homologues (but not SiaA from Neisseria meningitidis) appear to be common in tetrameric organization. The revised two-base catalytic mechanism may involve His-125 (Glu-134 in GNE), as suggested by mutant activity analysis.

Crystal structures of Staphylococcal SaeR reveal possible DNA-binding modes.

Two-component system SaeRS of Staphylococcus regulates virulence factor expression through phosphorylation of the DNA-binding regulator SaeR by the sensor histidine kinase SaeS. Here crystal structures of the DNA-binding domain (DBD) of SaeR from two Staphylococcal species Staphylococcus epidermidis and Staphylococcus aureus were determined and showed similar folds. Analyzing the DNA binding activity of three mutants of SeSaeR, we observed that Thr217 is important in binding to the phosphate group of DNA and Trp219 may interact with the base pairs. Additionally, the tandem arrangement of DBD may represent a possible way for SaeR oligomerization on DNA.

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme.

Structure and mechanism of an antibiotics-synthesizing 3-hydroxykynurenine C-methyltransferase.

Streptosporangium sibiricum SibL catalyzes the methyl transfer from S-adenosylmethionine (SAM) to 3-hydroxykynurenine (3-HK) to produce S-adenosylhomocysteine (SAH) and 3-hydroxy-4-methyl-kynurenine for sibiromycin biosynthesis. Here, we present the crystal structures of apo-form Ss-SibL, Ss-SibL/SAH binary complex and Ss-SibL/SAH/3-HK ternary complex. Ss-SibL is a homodimer. Each subunit comprises a helical N-terminal domain and a Rossmann-fold C-terminal domain. SAM (or SAH) binding alone results in domain movements, suggesting a two-step catalytic cycle. Analyses of the enzyme-ligand interactions and further mutant studies support a mechanism in which Tyr134 serves as the principal base in the transferase reaction of methyl group from SAM to 3-HK.

Crystal structure of a conserved hypothetical protein MJ0927 from Methanocaldococcus jannaschii reveals a novel quaternary assembly in the Nif3 family.

A Nif3 family protein of Methanocaldococcus jannaschii, MJ0927, is highly conserved from bacteria to humans. Although several structures of bacterial Nif3 proteins are known, no structure representing archaeal Nif3 has yet been reported. The crystal structure of Methanocaldococcus jannaschii MJ0927 was determined at 2.47 Å resolution to understand the structural differences between the bacterial and archaeal Nif3 proteins. Intriguingly, MJ0927 is found to adopt an unusual assembly comprising a trimer of dimers that forms a cage-like architecture. Electrophoretic mobility-shift assays indicate that MJ0927 binds to both single-stranded and double-stranded DNA. Structural analysis of MJ0927 reveals a positively charged region that can potentially explain its DNA-binding capability. Taken together, these data suggest that MJ0927 adopts a novel quartenary architecture that could play various DNA-binding roles in Methanocaldococcus jannaschii.