title: Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage.
abstract: Asparaginyl endopeptidase (AEP) is an endo/lysosomal cysteine endopeptidase with a preference for an asparagine residue at the P1 site and plays an important role in the maturation of toll-like receptors 3/7/9. AEP is known to undergo autoproteolytic maturation at acidic pH for catalytic activation. Here, we describe crystal structures of the AEP proenzyme and the mature forms of AEP. Structural comparisons between AEP and caspases revealed similarities in the composition of key residues and in the catalytic mechanism. Mutagenesis studies identified N44, R46, H150, E189, C191, S217/S218 and D233 as residues that are essential for the cleavage of the peptide substrate. During maturation, autoproteolytic cleavage of AEP's cap domain opens up access to the active site on the core domain. Unexpectedly, an intermediate autoproteolytic maturation stage was discovered at approximately pH 4.5 in which the partially activated AEP could be reversed back to its proenzyme form. This unique feature was confirmed by the crystal structure of AEPpH4.5 (AEP was matured at pH 4.5 and crystallized at pH 8.5), in which the broken peptide bonds were religated and the structure was transformed back to its proenzyme form. Additionally, the AEP inhibitor cystatin C could be digested by the fully activated AEP, but could not be digested by activated cathepsins. Thus, we demonstrate for the first time that cystatins may regulate the activity of AEP through substrate competition for the active site.
authors.affiliation: 1] National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Zhao
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Tian
authors.initials: T
authors.lastname: Hua
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Crowley
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Ru
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Ni
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Neil
authors.initials: N
authors.lastname: Shaw
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Lianying
authors.initials: L
authors.lastname: Jiao
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Qu
authors.affiliation: Physics Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Hung
authors.affiliation: iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.affiliation: Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Keqiang
authors.initials: K
authors.lastname: Ye
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Songying
authors.initials: S
authors.lastname: Ouyang
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Genhong
authors.initials: G
authors.lastname: Cheng
authors.affiliation: 1] National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002403:Cathepsins / Q000378:metabolism; D018360:Crystallography, X-Ray; D055316:Cystatin C / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity; D000099240:Asparaginyl Endopeptidase
chemical_list: D002403:Cathepsins; D055316:Cystatin C; D003546:Cysteine Endopeptidases; D004792:Enzyme Precursors; D000099240:Asparaginyl Endopeptidase