Human transglutaminase 2 (TGase2) has various functions, including roles in various cellular processes such as apoptosis, development, differentiation, wound healing, and angiogenesis, and is linked ...to many diseases such as cancer. Although TGase2 has been considered an optimized drug target for the treatment of cancer, fibrosis, and neurodegenerative disorders, it has been difficult to generate TGase2-targeted drugs for clinical use because of the relatively flat and broad active site on TGase2. To design more specific and powerful inhibitors, detailed structural information about TGase2 complexed with various effector and inhibitor molecules is required. In this review, we summarized the current structural studies on TGase2, which will aid in designing drugs that can overcome the aforementioned limitations.
Peptidoglycan digestion by murein-degrading enzymes is a critical process in bacterial cell growth and/or cell division. The membrane-bound lytic murein transglycosylase A (MltA) is a ...murein-degrading enzyme; it catalyzes the cleavage of the β-1,4-glycosidic linkage between
N
-acetylmuramic acid and
N
-acetylglucosamine in peptidoglycans. Although substrate recognition and cleavage by MltA have been examined by previous structural and mutagenesis studies, the overall mechanism of MltA in conjunction with other functionally related molecules on the outer membrane of bacterial cells for peptidoglycan degradation has remained elusive. In this study, the crystal structure of MltA from the virulent human pathogen
Acinetobacter baumannii
is characterized and presented. The study indicated that MltA from
A. baumannii
forms homodimers via an extra domain which is specific to this species. Furthermore, the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane was modeled based on the structural and biochemical analysis.
CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal viruses have developed ...countermeasures, employing anti-CRISPR (Acr) proteins to counteract CRISPR-Cas systems. Despite the revolutionary impact of CRISPR-Cas systems on genome editing, concerns persist regarding potential off-target effects. Therefore, understanding the structural and molecular intricacies of diverse Acrs is crucial for elucidating the fundamental mechanisms governing CRISPR-Cas regulation. In this study, we present the structure of AcrIIA28 from Streptococcus phage Javan 128 and analyze its structural and functional features to comprehend the mechanisms involved in its inhibition of Cas9. Our current study reveals that AcrIIA28 is a metalloprotein that contains Zn2+ and abolishes the cleavage activity of Cas9 only from Streptococcus pyrogen (SpyCas9) by directly interacting with the REC3 domain of SpyCas9. Furthermore, we demonstrate that the AcrIIA28 interaction prevents the target DNA from being loaded onto Cas9. These findings indicate the molecular mechanisms underlying AcrIIA28-mediated Cas9 inhibition and provide valuable insights into the ongoing evolutionary battle between bacteria and phages.
Clustered regularly interspaced short palindromic repeats (CRISPRs)‐CRISPR‐associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and ...viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti‐CRISPRs (Acrs) that are capable of inhibiting the host CRISPR‐Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti‐CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.
PDB Code(s): 7F7P;
As a result of bacterial infection with viruses, bacteria have developed CRISPR-Cas as an adaptive immune system, which allows them to destroy the viral genetic material introduced via infection. ...However, viruses have also evolved to develop multiple anti-CRISPR proteins, which are capable of inactivating the CRISPR-Cas adaptive immune system to combat bacteria. In this study, we aimed to elucidate the molecular mechanisms associated with anti-CRISPR proteins by determining a high-resolution crystal structure (1.3 Å) of Type I-E anti-CRISPR protein called AcrIE2. Our structural analysis revealed that AcrIE2 was composed of unique folds comprising five antiparallel β-sheets (β1∼β5) surrounding one α-helix (α1) in the order, β2β1α1β5β4β3. Structural comparison of AcrIE2 with a structural homolog called AcrIF9 showed that AcrIE2 contained a long and flexible β4-β5 connecting loop and a distinct surface feature. These results indicated that the inhibitory mechanism of AcrIE2 might be different from that of AcrIF9. This unique structure of AcrIE2 indicates its special mode of CRISPR-Cas inhibitory activity. Therefore, this study helps us understand the diversity in the inhibitory mechanisms of Acr family.
•We provide the 1.3 Å high resolution structure of AcrIE2.•AcrIE2 comprises the following noble fold β2β1α1β5β4β3.•AcrIE2 has a long and flexible β4-β5 connecting loop and a distinct surface feature.•This structure of AcrIE2 indicates a special mode of CRISPR-Cas inhibitory activity.•The inhibitory mechanism of AcrIE2 might be different from that of AcrIF9.
CRISPR–Cas systems are well known host defense mechanisms that are conserved in bacteria and archaea. To counteract CRISPR–Cas systems, phages and viruses have evolved to possess multiple anti‐CRISPR ...(Acr) proteins that can inhibit the host CRISPR–Cas system via different strategies. The expression of acr genes is controlled by anti‐CRISPR‐associated (Aca) proteins that bind to an upstream promoter and regulate the expression of acr genes during transcription. Although the role of Aca as a transcriptional repressor has been demonstrated, the mechanism of action of Aca has not been determined. Here, the molecular mechanism underlying the Aca2‐mediated transcriptional control of acr genes was elucidated by determining the crystal structure of Aca2 from Oceanimonas smirnovii at a high resolution of 1.92 Å. Aca2 forms a dimer in solution, and dimerization of Aca2 is critical for specific promoter binding. The promoter‐binding strategy of dimeric Aca2 was also revealed by performing mutagenesis studies. The atomic structure of the Aca family shown in this study provides insights into the fine regulation of host defense and immune‐escape mechanisms and also demonstrates the conserved working mechanism of the Aca family.
In this study, the crystal structure of anti‐CRISPR‐associated 2 (Aca2) was solved and it was shown that Aca2 forms dimers in solution, which are critical for promoter binding; specific residues that are critical for DNA binding and their conservation were revealed. Since the mechanism of action is largely conserved among different Aca protein families, insights are provided into their structure and function.
•The codon-optimized CgtA43456 was produced in C41(DE3) co-expressed with GroEL/ES.•The codon-optimized CgtB11168 was produced in C41(DE3).•The enzymes were obtained with ∼75–78 % purity using ...immobilized-metal affinity chromatography.•The specific activities of CgtA43456 and CgtB11168 were 21 and 16 mU/mg, respectively.
The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (β-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (β-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli–preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75–78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside–related oligosaccharides.
Abstract
CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages ...and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.
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.
•We solved the crystal structure of TRAF5 from Notathenia coriiceps.•We explained cold-adaptivity based on the structure of ncTRAF5.•We revealed trimeric structure as a functional unit of the ncTRAF5.•Finally, we suggested the tentative model of ncTRAF5 interactions with its various receptors.
This study determined the 1.2 Å high‐resolution crystal structure of AcrIF9 for the purpose of understanding the molecular basis underlying its anti‐CRISPR function. This high‐resolution crystal ...structure was compared with the identified structure of the cryo‐electron microscopy structure of AcrIF9 associated with the cascade complex to analyze feature similarities and differences.
Prokaryotic adaptive immunity by CRISPR‐Cas systems, which confer resistance to foreign genetic elements, has been used by bacteria to combat viruses. To cope, viruses evolved multiple anti‐CRISPR proteins, which can inhibit system function through various mechanisms. Although the structures and mechanisms of several anti‐CRISPR proteins have been elucidated, those of the AcrIF9 family have not yet been identified. To understand the molecular basis underlying AcrIF9 anti‐CRISPR function, we determined the 1.2 Å crystal structure of AcrIF9. Structural and biochemical studies showed that AcrIF9 exists in monomeric form in solution and can directly interact with DNA using a positively charged cleft. Based on analysis of the structure, we suggest part of the anti‐CRISPR molecular mechanism by AcrIF9.
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