Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-i...Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo- dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combi- nation of mutagenesis, biochemical, NMR, and small- angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubi- quitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiq- uitinating enzymes. Uch37AHb'Hc'KEKE, a truncation removal of the C-terminal extension region (residues 256- 329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270- 407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 com- plex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.展开更多
Endo-b-N-acetylglucosaminidases(ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have
Interferon gamma-inducible protein 16(IFI16)senses DNA in the cytoplasm and the nucleus by using two tandem hematopoietic interferon-inducible nuclear(HIN)domains,HINa and HINb,through the cooperative assembly of IFI1...Interferon gamma-inducible protein 16(IFI16)senses DNA in the cytoplasm and the nucleus by using two tandem hematopoietic interferon-inducible nuclear(HIN)domains,HINa and HINb,through the cooperative assembly of IFI16 filaments on double-stranded DNA(dsDNA).The role of HINa in sensing DNA is not clearly understood.Here,we describe the crystal structure of the HINa domain in complex with DNA at 2.55A°resolution and provide the first insight into the mode of DNA binding by the HINa domain.The structure reveals the presence of two oligosaccharide/nucleotide-binding(OB)folds with a unique DNA-binding surface.HINa uses loop L45 of the canonical OB2 fold to bind to the DNA backbone.The dsDNA is recognized as two single strands of DNA.Interestingly,deletion of HINb compromises the ability of IFI16 to induce IFN-b,while HINa mutants impaired in DNAbinding enhance the production of IFN-b.These results shed light on the roles of IFI16 HIN domains in DNA recognition and innate immune responses.展开更多
Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell.Amongst the spillage,the secondary messenger molecule,cyclic dimeric guanosine monophosphate(c di-GMP),was rece...Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell.Amongst the spillage,the secondary messenger molecule,cyclic dimeric guanosine monophosphate(c di-GMP),was recently shown to be bound by stimulator of interferon genes(STING).Binding of c di-GMP by STING activates the Tank Binding Kinase(TBK1)mediated signaling cascades that galvanize the body’s defenses for elimi-nation of the pathogen.In addition to c di-GMP,STING has also been shown to function in innate immune re-sponses against pathogen associated molecular pat-terns(PAMPs)originating from the DNA or RNA of pathogens.The pivotal role of STING in host defense is exemplified by the fact that STING-/-mice die upon infection by HSV-1.Thus,STING plays an essential role in innate immune responses against pathogens.This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the im-mune defenses against invading microbes.Similarly,STING could be targeted for mitigating the inflamma-tory responses augmented by the innate immune sys-tem.This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in de-lineating the mechanism of STING-mediated responses.展开更多
Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobac- terium tuberculosis (Mtb). Consequently, stalling dehy-dration cures tuberculosis (TB). Clinically us...Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobac- terium tuberculosis (Mtb). Consequently, stalling dehy-dration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxy- acyI-ACP dehydratase HadAB complex. How this inhi- bition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon muta- tion to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site, The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs,展开更多
Acute intermittent porphyria (AIP), an inherited disease of heme biosynthesis, is one of the most common type of the porphyrias. Reduced activity of the enzyme
Sometimes crystals cannot diffract X-rays beyond 3.0Åresolution due to the intrinsic flexibility associated with the protein.Low resolution diffraction data not only pose a challenge to structure determination,bu...Sometimes crystals cannot diffract X-rays beyond 3.0Åresolution due to the intrinsic flexibility associated with the protein.Low resolution diffraction data not only pose a challenge to structure determination,but also hamper interpretation of mechanistic details.Crystals of a 25.6 kDa non-Pfam,hypothetical protein,PF2046,diffracted X-rays to 3.38Åresolution.A combination of SeMet derived heavy atom positions with multiple cycles of B-factor sharpening,multi-crystal averaging,restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5(R_(free)=24.7).The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers.Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.展开更多
基金This work was supported by the National Basic Research Program (973 Program) (Nos. 2014CB910400 and 2013CB911103), the Ministry of Health of China (Grant No. 2013ZX10004-602), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant No. 2014BAI07B02) and the National Natural Science Foundation of China (Grant Nos. 31330019, 31200559). We would like to thank Dr. Li-Qin Li from the Institute of High Energy Physics, CAS and Professor Robert E. Cohen for the gen- erous gift of the hUch37 (C88A) plasmid and Xiaoxia Yu and Yu- anyuan Chen at the Protein Science Core Facility of IBP for their technical help with the AUC and SPR experiments. The authors would also like to thank the staff at beamline BL13.3.1 at ALS for their technical support with the SAXS data collection. BL13.3.1 is supported in part by the DOE program Inte- grated Diffraction Analysis Technologies (IDAT) and the DOE pro- gram Molecular Assemblies Genes and Genomics Integrated Efficiently (MAGGIE) under Contract Number DE-AC02-05CH11231 with the DOE. The ALS is supported by the Director, Office of Sci- ence, Office of Basic Energy Sciences of the DOE under Contract No. DE-AC02-05CH11231. Use of the Advanced Photon Source, an Office of the Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02- 06CHl1357. BioCAT was supported by grants from the National Center for Research Resources (2P41RR008630-17) and the National Institute of General Medical Sciences (9 P41 GM103622- 17) from the National Institutes of Health. The authors would like to thank the staff at 121D and 181D for the setup support.
文摘Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo- dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combi- nation of mutagenesis, biochemical, NMR, and small- angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubi- quitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiq- uitinating enzymes. Uch37AHb'Hc'KEKE, a truncation removal of the C-terminal extension region (residues 256- 329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270- 407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 com- plex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
文摘Endo-b-N-acetylglucosaminidases(ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have
基金supported by the National Natural Science Foundation of China(grant no.31570875,31330019,31200559,and 81590761)the Ministry of Science and Technology of China(grant no.2014CB910400 and 2013CB911103)+2 种基金the National Science and Technology Major Project of China(grant no.2013ZX10004-602)the Beijing Nova Program(grant no.Z141102001814020)Youth Innovation Promotion Association CAS,and the special project of Ebola virus research from the president foundation of Chinese Academy of Sciences.
文摘Interferon gamma-inducible protein 16(IFI16)senses DNA in the cytoplasm and the nucleus by using two tandem hematopoietic interferon-inducible nuclear(HIN)domains,HINa and HINb,through the cooperative assembly of IFI16 filaments on double-stranded DNA(dsDNA).The role of HINa in sensing DNA is not clearly understood.Here,we describe the crystal structure of the HINa domain in complex with DNA at 2.55A°resolution and provide the first insight into the mode of DNA binding by the HINa domain.The structure reveals the presence of two oligosaccharide/nucleotide-binding(OB)folds with a unique DNA-binding surface.HINa uses loop L45 of the canonical OB2 fold to bind to the DNA backbone.The dsDNA is recognized as two single strands of DNA.Interestingly,deletion of HINb compromises the ability of IFI16 to induce IFN-b,while HINa mutants impaired in DNAbinding enhance the production of IFN-b.These results shed light on the roles of IFI16 HIN domains in DNA recognition and innate immune responses.
基金supported by the National Natural Science Foundation of China(Grant No.31070660)the Ministry of Science and Technology of China(No.2009DFB30310).
文摘Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell.Amongst the spillage,the secondary messenger molecule,cyclic dimeric guanosine monophosphate(c di-GMP),was recently shown to be bound by stimulator of interferon genes(STING).Binding of c di-GMP by STING activates the Tank Binding Kinase(TBK1)mediated signaling cascades that galvanize the body’s defenses for elimi-nation of the pathogen.In addition to c di-GMP,STING has also been shown to function in innate immune re-sponses against pathogen associated molecular pat-terns(PAMPs)originating from the DNA or RNA of pathogens.The pivotal role of STING in host defense is exemplified by the fact that STING-/-mice die upon infection by HSV-1.Thus,STING plays an essential role in innate immune responses against pathogens.This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the im-mune defenses against invading microbes.Similarly,STING could be targeted for mitigating the inflamma-tory responses augmented by the innate immune sys-tem.This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in de-lineating the mechanism of STING-mediated responses.
文摘Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobac- terium tuberculosis (Mtb). Consequently, stalling dehy-dration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxy- acyI-ACP dehydratase HadAB complex. How this inhi- bition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon muta- tion to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site, The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs,
文摘Acute intermittent porphyria (AIP), an inherited disease of heme biosynthesis, is one of the most common type of the porphyrias. Reduced activity of the enzyme
基金This work was funded by the Ministry of Science and Technology of China(Grant Nos.2006AA02A316,2009DFB30310 and 2006CB910901)the National Natural Science Foundation of China(Grants Nos.30670427 and 30721003)+1 种基金the Ministry of Health of China(Grant No.2008ZX10404)CAS Research Grant(No.KSCX2-YW-R-127 and INFO-115-D01-2009).
文摘Sometimes crystals cannot diffract X-rays beyond 3.0Åresolution due to the intrinsic flexibility associated with the protein.Low resolution diffraction data not only pose a challenge to structure determination,but also hamper interpretation of mechanistic details.Crystals of a 25.6 kDa non-Pfam,hypothetical protein,PF2046,diffracted X-rays to 3.38Åresolution.A combination of SeMet derived heavy atom positions with multiple cycles of B-factor sharpening,multi-crystal averaging,restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5(R_(free)=24.7).The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers.Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.