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ABSTRACT: The energy sensor AMP-activated protein kinase (AMPK) is a heterotrimeric complex that is allosterically activated by AMP binding to the γ subunit. Cocrystal structures of the mammalian AMPK core reveal occlusion of nucleotide-binding site 3 of the γ subunit in the presence of ATP. However, site 3 is occupied in the presence of AMP. Mutagenesis studies indicate that sites 3 and 4 are important for AMPK allosteric activation.
Nature Structural & Molecular Biology 06/2012; 19(7):716-8. · 12.71 Impact Factor
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ABSTRACT: p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.
Structure 12/2011; 19(12):1752-61. · 6.35 Impact Factor
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ABSTRACT: Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent
signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms
of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively
between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation
mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine,
Thr402, in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation
suggest that autophosphorylation of Thr402 in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr402 to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr402 in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the
study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would
be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.
Journal of Biological Chemistry 01/2011; 286(4):2689-2695. · 4.77 Impact Factor
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ABSTRACT: Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine, Thr(402), in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation suggest that autophosphorylation of Thr(402) in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr(402) to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr(402) in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.
Journal of Biological Chemistry 01/2011; 286(4):2689-95. · 4.77 Impact Factor
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Xiang Gao, Jue Wang,
Da-Qi Yu,
Fei Bian,
Bin-Bin Xie,
Xiu-Lan Chen,
Bai-Cheng Zhou,
Lu-Hua Lai,
Zhi-Xin Wang,
Jia-Wei Wu,
Yu-Zhong Zhang
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ABSTRACT: Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent T(m) of the autoprocessed complex increases 20 °C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.
Proceedings of the National Academy of Sciences 09/2010; 107(41):17569-74. · 9.68 Impact Factor
