Owen, GRStoychev, Stoyan HAchilonu, IDirr, HW2015-03-122015-03-122014-10Owen, G.R, Stoychev, S, Achilonu, I and Dirr, H.W. 2014. Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation. Journal of Molecular Biology. Vol. 426(21), pp. 3569–35890022-2836http://hdl.handle.net/10204/7883https://www.sciencedirect.com/science/article/pii/S0022283614004628https://doi.org/10.1016/j.jmb.2014.08.019Copyright: 2014 Elsevier. This is the pre-print/post-print version of the work. The definitive version is published in the Journal of Molecular Biology. Vol. 426(21), pp. 3569–3589. Due to copyright restrictions, the attached PDF file only contains the abstract of the full text item. For access to the full text item, please consult the publisher's websiteMany 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 JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1ß1 using the ATP analogue, 5'-adenylyl-imidodiphosphate (AMP-PNP). JNK1ß1 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 JNK1ß1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1ß1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1ß1 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 JNK1ß1 is regulated.enc-Jun N-terminal kinaseMAP kinasesHydrogen exchangeMass spectrometryProtein dynamicsArticleOwen, G., Stoychev, S. H., Achilonu, I., & Dirr, H. (2014). Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation. http://hdl.handle.net/10204/7883Owen, GR, Stoyan H Stoychev, I Achilonu, and HW Dirr "Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation." (2014) http://hdl.handle.net/10204/7883Owen G, Stoychev SH, Achilonu I, Dirr H. Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation. 2014; http://hdl.handle.net/10204/7883.TY - Article AU - Owen, GR AU - Stoychev, Stoyan H AU - Achilonu, I AU - Dirr, HW AB - 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 JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1ß1 using the ATP analogue, 5'-adenylyl-imidodiphosphate (AMP-PNP). JNK1ß1 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 JNK1ß1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1ß1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1ß1 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 JNK1ß1 is regulated. DA - 2014-10 DB - ResearchSpace DP - CSIR KW - c-Jun N-terminal kinase KW - MAP kinases KW - Hydrogen exchange KW - Mass spectrometry KW - Protein dynamics LK - https://researchspace.csir.co.za PY - 2014 SM - 0022-2836 T1 - Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation TI - Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation UR - http://hdl.handle.net/10204/7883 ER -