title: Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).
abstract: JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications.
authors.affiliation: Departments of Therapeutic Discovery, Amgen Inc., South San Francisco, California 94080.
authors.forename: Xiaoshan
authors.initials: X
authors.lastname: Min
authors.affiliation: the Institute of Biomedical Technology, University of Tampere, 33014 Tampere, Finland.
authors.forename: Daniela
authors.initials: D
authors.lastname: Ungureanu
authors.affiliation: Departments of Inflammation, Amgen Inc., South San Francisco, California 94080.
authors.forename: Sarah
authors.initials: S
authors.lastname: Maxwell
authors.affiliation: the School of Medicine, University of Tampere, 33014 Tampere, Finland.
authors.forename: Henrik
authors.initials: H
authors.lastname: Hammarén
authors.affiliation: Departments of Therapeutic Discovery, Amgen Inc., South San Francisco, California 94080.
authors.forename: Steve
authors.initials: S
authors.lastname: Thibault
authors.affiliation: the Institute of Biomedical Technology, University of Tampere, 33014 Tampere, Finland.
authors.forename: Ellin-Kristina
authors.initials: EK
authors.lastname: Hillert
authors.affiliation: Departments of Therapeutic Discovery, Amgen Inc., South San Francisco, California 94080.
authors.forename: Merrill
authors.initials: M
authors.lastname: Ayres
authors.affiliation: Departments of Inflammation, Amgen Inc., South San Francisco, California 94080.
authors.forename: Brad
authors.initials: B
authors.lastname: Greenfield
authors.affiliation: Departments of Therapeutic Discovery, Amgen Inc., South San Francisco, California 94080.
authors.forename: John
authors.initials: J
authors.lastname: Eksterowicz
authors.affiliation: Departments of Inflammation, Amgen Inc., South San Francisco, California 94080.
authors.forename: Chris
authors.initials: C
authors.lastname: Gabel
authors.affiliation: Departments of Therapeutic Discovery, Amgen Inc., South San Francisco, California 94080.
authors.forename: Nigel
authors.initials: N
authors.lastname: Walker
authors.affiliation: the School of Medicine, University of Tampere, 33014 Tampere, Finland; Department of Clinical Hematology, Tampere University Hospital, 33520 Tampere, Finland. Electronic address: olli.silvennoinen@uta.fi.
authors.forename: Olli
authors.initials: O
authors.lastname: Silvennoinen
authors.affiliation: Departments of Therapeutic Discovery, Amgen Inc., South San Francisco, California 94080. Electronic address: zwang@amgen.com.
authors.forename: Zhulun
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D053613:Janus Kinase 1 / Q000737:chemistry; D053614:Janus Kinase 2 / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D053634:TYK2 Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; C507904:JAK1 protein, human; C507924:JAK2 protein, human; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D053634:TYK2 Kinase; C070139:TYK2 protein, human
keywords: ATP binding; JAK tyrosine kinase; Janus kinase (JAK); TYK2; X-ray crystallography; crystal structure; protein kinase; pseudokinase domain; structural biology; structure-function