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.
Transglutaminase 2 (TG2) is a Ca 2+ -dependent enzyme, which regulates various cellular processes by catalyzing protein crosslinking or polyamination. Intracellular TG2 is activated and inhibited by Ca 2+ and GTP binding, respectively. Although aberrant TG2 activation has been implicated in the pathogenesis of diverse diseases, including cancer and degenerative and fibrotic diseases, the structural basis for the regulation of TG2 by Ca 2+ and GTP binding is not fully understood.Here, we produced and analyzed a Ca 2+ -containing TG2 crystal, and identified two glutamate residues, E437 and E539, as Ca 2+ -binding sites. The enzymatic analysis of the mutants revealed that Ca 2+ binding to these sites is required for the transamidase activity of TG2. Interestingly, we found that magnesium (Mg 2+ ) competitively binds to the E437 and E539 residues. The Mg 2+ binding to these allosteric sites enhances the GTP binding/hydrolysis activity but inhibits transamidase activity. Furthermore, HEK293 cells transfected with mutant TG2 exhibited higher transamidase activity than cells with wild-type TG2. Cells with wild-type TG2 showed an increase in transamidase activity under Mg 2+ -depleted conditions, whereas cells with mutant TG2 were unaffected. These results indicate that E437 and E539 are Ca 2+ -binding sites contributing to the reciprocal regulation of transamidase and GTP binding/hydrolysis activities of TG2 through competitive Mg 2+ binding.
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.
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