title: Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1β1 provide insight into its mechanisms of activation.
abstract: Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen-deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1β1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1β1 using the ATP analogue, 5'-adenylyl-imidodiphosphate (AMP-PNP). JNK1β1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1β1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1β1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1β1 is regulated.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Gavin R
authors.initials: GR
authors.lastname: Owen
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria 0001, South Africa.
authors.forename: Stoyan
authors.initials: S
authors.lastname: Stoychev
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa. Electronic address: heinrich.dirr@wits.ac.za.
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D020935:MAP Kinase Signaling System; D013058:Mass Spectrometry; D048057:Mitogen-Activated Protein Kinase 10 / Q000737:chemistry*; D048055:Mitogen-Activated Protein Kinase 8 / Q000737:chemistry*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D009711:Nucleotides; D012997:Solvents; D006859:Hydrogen; D000255:Adenosine Triphosphate; D014508:Urea; D048057:Mitogen-Activated Protein Kinase 10; D048055:Mitogen-Activated Protein Kinase 8
keywords: MAP kinases; c-Jun N-terminal kinase; hydrogen exchange; mass spectrometry; protein dynamics