Structural basis of Cas3 activation in type I-C CRISPR-Cas system.

CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication.

Interdomain flexibility and putative active site was revealed by crystal structure of MltG from Acinetobacter baumannii.

MltG, positioned within the inner membrane of bacteria, functions as a lytic transglycosylase (LT) essential for integrating into the cell wall by cleaving the newly synthesized glycan strand, emphasizing its critical involvement in bacterial cell wall biosynthesis and remodeling. Current study reported the first structure of MltG family of LT. We have elucidated the structure of MltG from Acinetobacter baumannii (abMltG), a formidable superbug renowned for its remarkable antibiotic resistance. Our structural and biochemical investigations unveiled the presence of a flexible peptidoglycan (PG)-binding domain (PGD) within MltG family, which exists as a monomer in solution. Furthermore, we delineated the putative active site of abMltG via a combination of structural analysis and sequence comparison. This discovery enhances our comprehension of the transglycosylation process mediated by the MltG family, offering insights that could inform the development of novel antibiotics tailored to combat A. baumannii.

Novel structure of the anti-CRISPR protein AcrIE3 and its implication on the CRISPR-Cas inhibition.

As a response to viral infections, bacteria have evolved the CRISPR-Cas system as an adaptive immune mechanism, enabling them to target and eliminate viral genetic material introduced during infection. However, viruses have also evolved mechanisms to counteract this bacterial defense, including anti-CRISPR proteins, which can inactivate the CRISPR-Cas adaptive immune system, thus aiding the viruses in their survival and replication within bacterial hosts. In this study, we establish the high-resolution crystal structure of the Type IE anti-CRISPR protein, AcrIE3. Our structural examination showed that AcrIE3 adopts a helical bundle fold comprising four α-helices, with a notably extended loop at the N-terminus. Additionally, surface analysis of AcrIE3 revealed the presence of three acidic regions, which potentially play a crucial role in the inhibitory function of this protein. The structural information we have elucidated for AcrIE3 will provide crucial insights into fully understanding its inhibitory mechanism. Furthermore, this information is anticipated to be important for the application of the AcrIE family in genetic editing, paving the way for advancements in gene editing technologies.

Molecular basis of neurodevelopmental disorders caused by pathogenic variants of PIDD.

PIDDosome formation followed by caspase-2 activation is critical for genotoxic stress-induced apoptotic cell death. Failure of proper caspase-2 activation causes a neurodevelopmental disorder and intellectual disability. R815W, R862W, and Q863stop mutations in p53-induced protein with a death domain (PIDD), a component of the PIDDosome, also lead to this disorder. However, the molecular mechanisms underlying this pathogenesis remain elusive. In this study, we analyzed the molecular mechanisms underlying the pathogenesis of the PIDD DD pathogenic variants R815W, R862W, and Q863stop. We determined that these mutations prevented the interaction between PIDD and RIP-associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD), a molecule that mediates PIDDosome formation. The disruption of this interaction affects PIDDosome formation and caspase-2 activation.

Structure of YdjH from Acinetobacter baumannii revealed an active site of YdjH family sugar kinase.

Bacterial sugar kinase is a central enzyme for proper sugar degradation in bacteria, essential for survival and growth. Therefore, this enzyme family is a primary target for antibacterial drug development, with YdjH most preferring to phosphorylate higher-order monosaccharides with a carboxylate terminus. Sugar kinases express diverse specificity and functions, making specificity determination of this family a prominent issue. This study examines the YdjH crystal structure from Acinetobacter baumannii (abYdjH), which has an exceptionally high antibiotic resistance and is considered a superbug. Our structural and biochemical study revealed that abYdjH has a widely open lid domain and is a solution dimer. In addition, the putative active site of abYdjH was determined based on structural analysis, sequence comparison, and in silico docking. Finally, we proposed the active site-forming residues that determine various sugar specificities from abYdjH. This study contributes towards a deeper understanding of the phosphorylation process and bacterial sugar metabolism of YdjH family to design the next-generation antibiotics for targeting A. baumannii.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Structural and biochemical characterization of TRAF5 from Notothenia coriiceps and its implications in fish immune cell signaling.

Conserved immune cell signaling in fish was recently highlighted by the identification of various immune cell signaling molecules. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are critical adaptor molecules in immune cell signaling and contain E3 ubiquitin ligase activity. Here, we report the first crystal structure of the TRAF5 TRAF domain from the black rockcod (Notothenia coriiceps; ncTRAF5). Our structure revealed both similarities and differences with mammalian TRAF5. Structural and biochemical analyses indicated that ncTRAF5 forms a functional trimer unit in solution, with a structural flexibility that might be critical for imparting resistance to cold temperature-induced stress. We also found conserved surface residues on ncTRAF5 that might be critical binding hot spots for interaction with various receptors.

Structural insights into the enzyme specificity of a novel ω-transaminase from the thermophilic bacterium Sphaerobacter thermophilus.

Transaminases are pyridoxal 5'-phosphate-dependent enzymes that reversibly catalyze transamination reactions from an amino group donor substrate to an amino group acceptor substrate. ω-Transaminases (ωTAs) utilize compounds with an amino group not at α-carbon position as their amino group donor substrates. Recently, a novel ωTA with broad substrate specificity and high thermostability from the thermophilic bacterium Sphaerobacter thermophilus (St-ωTA) has been reported. Although St-ωTA has been biochemically characterized, little is known about its determinants of substrate specificity. In the present study, we determined the crystal structure of St-ωTA at 1.9 Šresolution to clarify in detail its mechanism of substrate recognition. The structure of St-ωTA revealed that it has a voluminous active site resulting from the unique spatial arrangement of residues comprising its active site. In addition, our molecular docking simulation results suggest that substrate compounds may bind to active site residues via electrostatic interactions or hydrophobic interactions that can be induced by subtle rearrangements of active site residues. On the basis of these structural analyses, we propose a plausible working model of the enzymatic mechanism of St-ωTA. Our results provide profound structural insights into the substrate specificity of St-ωTA and extend the boundaries of knowledge of TAs.

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3.

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.

Identification and analysis of dominant negative mutants of RIP1 DD that disrupt RIPoptosome core formation.

The RIPoptosome, composed of RIP1 and caspase-8, plays an important role in the regulation of apoptosis and necroptosis; however, the mechanism of complex formation by oligomerization and how the caspase-activating process and necroptosis are mediated by the formation of the RIPoptosome is not well-understood. This study revealed that the assembly mechanism of the RIPoptosome core is dependent on salt concentration and not on pH and time. In addition, we demonstrated that three RIP1 mutations, E626K, M637K, and S657K, have dominant negative effects. These dominant negative mutations in RIP1 may have potential applications in therapeutic intervention.

Structural basis of IRGB10 oligomerization by GTP hydrolysis.

Immunity-related GTPase B10 (IRGB10) is a crucial member of the interferon (IFN)-inducible GTPases and plays a vital role in host defense mechanisms. Following infection, IRGB10 is induced by IFNs and functions by liberating pathogenic ligands to activate the inflammasome through direct disruption of the pathogen membrane. Despite extensive investigation into the significance of the cell-autonomous immune response, the precise molecular mechanism underlying IRGB10-mediated microbial membrane disruption remains elusive. Herein, we present two structures of different forms of IRGB10, the nucleotide-free and GppNHp-bound forms. Based on these structures, we identified that IRGB10 exists as a monomer in nucleotide-free and GTP binding states. Additionally, we identified that GTP hydrolysis is critical for dimer formation and further oligomerization of IRGB10. Building upon these observations, we propose a mechanistic model to elucidate the working mechanism of IRGB10 during pathogen membrane disruption.

The structure of AcrIC9 revealing the putative inhibitory mechanism of AcrIC9 against the type IC CRISPR-Cas system.

CRISPR-Cas systems are known to be part of the bacterial adaptive immune system that provides resistance against intruders such as viruses, phages and other mobile genetic elements. To combat this bacterial defense mechanism, phages encode inhibitors called Acrs (anti-CRISPR proteins) that can suppress them. AcrIC9 is the most recently identified member of the AcrIC family that inhibits the type IC CRISPR-Cas system. Here, the crystal structure of AcrIC9 from Rhodobacter capsulatus is reported, which comprises a novel fold made of three central antiparallel ß-strands surrounded by three α-helixes, a structure that has not been detected before. It is also shown that AcrIC9 can form a dimer via disulfide bonds generated by the Cys69 residue. Finally, it is revealed that AcrIC9 directly binds to the type IC cascade. Analysis and comparison of its structure with structural homologs indicate that AcrIC9 belongs to DNA-mimic Acrs that directly bind to the cascade complex and hinder the target DNA from binding to the cascade.

Molecular basis of apoptotic DNA fragmentation by DFF40.

Although the functions of CIDE domain-containing proteins, including DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, in apoptotic DNA fragmentation and lipid homeostasis have been studied extensively in mammals, the functions of four CIDE domain-containing proteins identified in the fly, namely DREP1, 2, 3, and 4, have not been explored much. Recent structural study of DREP4, a fly orthologue of mammalian DFF40 (an endonuclease involved in apoptotic DNA fragmentation), showed that the CIDE domain of DREP4 (and DFF40) forms filament-like assembly, which is critical for the corresponding function. The current study aimed to investigate the mechanism of filament formation of DREP4 CIDE and to characterize the same. DREP4 CIDE was shown to specifically bind to histones H1 and H2, an event important for the nuclease activity of DREP4. Based on the current experimental results, we proposed the mechanism underlying the process of apoptotic DNA fragmentation.

Helical filament structure of the DREP3 CIDE domain reveals a unified mechanism of CIDE-domain assembly.

The cell-death-inducing DFF45-like effector (CIDE) domain is a protein-interaction module comprising ∼80 amino acids and was initially identified in several apoptotic nucleases and their regulators. CIDE-domain-containing proteins were subsequently identified among proteins involved in lipid metabolism. Given the involvement of CIDE-domain-containing proteins in cell death and lipid homeostasis, their structure and function have been intensively studied. Here, the head-to-tail helical filament structure of the CIDE domain of DNA fragmentation factor-related protein 3 (DREP3) is presented. The helical filament structure was formed by opposing positively and negatively charged interfaces of the domain and was assembled depending on protein and salt concentrations. Although conserved filament structures are observed in CIDE family members, the structure elucidated in this study and its comparison with previous structures indicated that the size and the number of molecules used in one turn vary. These findings suggest that this charged-surface-based head-to-tail helical filament structure represents a unified mechanism of CIDE-domain assembly and provides insight into the function of various forms of the filament structure of the CIDE domain in higher-order assembly for apoptotic DNA fragmentation and control of lipid-droplet size.

Heterogeneous multimeric structure of isocitrate lyase in complex with succinate and itaconate provides novel insights into its inhibitory mechanism.

During the glyoxylate cycle, isocitrate lyases (ICLs) catalyze the lysis of isocitrate to glyoxylate and succinate. Itaconate has been reported to inhibit an ICL from Mycobacterium tuberculosis (tbICL). To elucidate the molecular mechanism of ICL inhibition, we determined the crystal structure of tbICL in complex with itaconate. Unexpectedly, succinate and itaconate were found to bind to the respective active sites in the dimeric form of tbICL. Our structure revealed the active site architecture as an open form, although the substrate and inhibitor were bound to the active sites. Our findings provide novel insights into the conformation of tbICL upon its binding to a substrate or inhibitor, along with molecular details of the inhibitory mechanism of itaconate.

Molecular basis of IRGB10 oligomerization and membrane association for pathogen membrane disruption.

Immunity-related GTPase B10 (IRGB10) belongs to the interferon (IFN)-inducible GTPases, a family of proteins critical to host defense. It is induced by IFNs after pathogen infection, and plays a role in liberating pathogenic ligands for the activation of the inflammasome by directly disrupting the pathogen membrane. Although IRGB10 has been intensively studied owing to its functional importance in the cell-autonomous immune response, the molecular mechanism of IRGB10-mediated microbial membrane disruption is still unclear. In this study, we report the structure of mouse IRGB10. Our structural study showed that IRGB10 bound to GDP forms an inactive head-to-head dimer. Further structural analysis and comparisons indicated that IRGB10 might change its conformation to activate its membrane-binding and disruptive functions. Based on this observation, we propose a model of the working mechanism of IRGB10 during pathogen membrane disruption.

Wide-open conformation of UDP-MurNc-tripeptide ligase revealed by the substrate-free structure of MurE from Acinetobacter baumannii.

MurE ligase catalyzes the attachment of meso-diaminopimelic acid to the UDP-MurNAc-l -Ala-d -Glu using ATP and producing UDP-MurNAc-l -Ala-d -Glu-meso-A2 pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate-free structure of MurE from Acinetobacter baumannii, which is an antibiotic-resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide-open conformation and undergoes wide-open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next-generation antibiotics targeting MurE.

Crystal structure of the human NLRP9 pyrin domain reveals a bent N-terminal loop that may regulate inflammasome assembly.

Members of the NLR family pyrin domain containing (NLRPs) are pattern recognition receptors that participate in innate immunity. They form inflammasomes, which are platforms for caspase-1 recruitment and activation. The NLRP pyrin domain (PYD) is critical for the assembly of inflammasomes due to its ability to mediate protein interactions. Despite intensive structural studies on inflammasomes with PYDs, the structure of the PYD of NLRP9-the least studied member of the family-remains unknown. Herein, we report the crystal structure of the human NLRP9 PYD at 2.1 Å resolution, which reveals a kinked N-terminal loop oriented toward the interior of the helical bundle. Based on our findings, we propose a regulatory role for the kinked N-terminal loop of NLRP9 PYD in inflammasome assembly.

Assembly of platforms for signal transduction in the new era: dimerization, helical filament assembly, and beyond.

Supramolecular organizing center (SMOC)-mediated signal transduction is an emerging concept in the field of signal transduction that is ushering in a new era. The formation of location-specific, higher-order SMOCs is particularly important for cell death and innate immune signaling processes. Several protein interaction domains, including the death domain (DD) superfamily and the CIDE domain, are representative mediators of SMOC assembly in cell death and innate immune signaling pathways. DD superfamily- and CIDE domain-containing proteins form SMOCs that activate various caspases and provide signaling scaffold platforms. These assemblies can lead to signal transduction and amplification during signaling events. In this review, we summarize recent findings on the molecular basis of DD superfamily- and CIDE domain-mediated SMOC formation.

Crystal structure and mutation analysis revealed that DREP2 CIDE forms a filament-like structure with features differing from those of DREP4 CIDE.

Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although our recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. In this study, we present the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE.