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The dynamical mechanism of auto-inhibition of AMP-activated protein kinase.

Peng C, Head-Gordon T - PLoS Comput. Biol. (2011)

Bottom Line: However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited.By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition.Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop.

View Article: PubMed Central - PubMed

Affiliation: MOE-Microsoft Key Laboratory for Intelligent Computing and Intelligent Systems, Department of Computer Science and Engineering, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
We use a novel normal mode analysis of an elastic network model drawn from configurations generated during microsecond all-atom molecular dynamics simulations to analyze the mechanism of auto-inhibition of AMP-activated protein kinase (AMPK). A recent X-ray and mutagenesis experiment (Chen, et al Nature 2009, 459, 1146) of the AMPK homolog S. Pombe sucrose non-fermenting 1 (SNF1) has proposed a new conformational switch model involving the movement of the kinase domain (KD) between an inactive unphosphorylated open state and an active or semi-active phosphorylated closed state, mediated by the autoinhibitory domain (AID), and a similar mutagenesis study showed that rat AMPK has the same auto-inhibition mechanism. However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited. By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition. Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop.

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Ribbon structures of the KD-AID fragment.(a) Comparison of the unphosphorylated KD-AID fragment (PDB Id: 3H4J, blue ribbons) that represents the open state with the homologous phosphorylated KD fragment (PDB Id: 3DAE, red ribbons) representing the closed state. (b) The N-terminal lobe (residues 27–114) and part of the C-terminal lobe (residues 115–175) of the KD domain are shown in orange, and are used to measure the interlobe conformational transition. The activation loop (residues 176–206) is shown in pink, with the rest of KD shown in green, and the AID fragment shown in red.
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pcbi-1002082-g001: Ribbon structures of the KD-AID fragment.(a) Comparison of the unphosphorylated KD-AID fragment (PDB Id: 3H4J, blue ribbons) that represents the open state with the homologous phosphorylated KD fragment (PDB Id: 3DAE, red ribbons) representing the closed state. (b) The N-terminal lobe (residues 27–114) and part of the C-terminal lobe (residues 115–175) of the KD domain are shown in orange, and are used to measure the interlobe conformational transition. The activation loop (residues 176–206) is shown in pink, with the rest of KD shown in green, and the AID fragment shown in red.

Mentions: Biochemical experiments have shown that the isolated full-length α-subunit and even the α1 isoform (residues 1–392) have little activity due to the presence of the conserved AID domain [10], [11]. Recently, an exciting X-ray crystallography study [12] has successfully crystallized an unphosphorylated fragment containing both KD and AID from Schizosaccharomyces pombe (PDB Id: 3H4J), and a phosphorylated fragment containing only KD from Saccharomyces cerevisiae (PDB Id: 3DAE), providing a static structural view of how AID inhibits the conformational transition of the N-terminal and C-terminal lobes in the KD domain to the functional closed state (Figure 1a) [13]. Mutagenesis of key residues of AID were found to restore catalytic function of the KD fragment, thereby isolating key residue interactions between these two domains. The same interface point mutations of the rat AMPK α1 subunit show exactly the same catalytic trends as the S.Pombe KD-AID fragment, which was further confirmed in the rat AMPK holoenzyme in that these same mutations both increase the catalytic activity and slow the dephosphorylation of the α-subunit, independent of AMP concentration. Based on the X-ray crystal structures and catalytic activity upon mutagenesis, the authors proposed a new conformational switch model for the regulatory mechanism of AMPK activity in which the interaction of AID with KD requires the latter to adopt a relatively open conformational form that is inactive. The eventual binding of AMP to the γ-subunit changes the interactions between the AID and KD, at present by an unknown molecular mechanism, to remove the inhibitory effect of AID to allow the interlobe conformational transition to the closed state.


The dynamical mechanism of auto-inhibition of AMP-activated protein kinase.

Peng C, Head-Gordon T - PLoS Comput. Biol. (2011)

Ribbon structures of the KD-AID fragment.(a) Comparison of the unphosphorylated KD-AID fragment (PDB Id: 3H4J, blue ribbons) that represents the open state with the homologous phosphorylated KD fragment (PDB Id: 3DAE, red ribbons) representing the closed state. (b) The N-terminal lobe (residues 27–114) and part of the C-terminal lobe (residues 115–175) of the KD domain are shown in orange, and are used to measure the interlobe conformational transition. The activation loop (residues 176–206) is shown in pink, with the rest of KD shown in green, and the AID fragment shown in red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140967&req=5

pcbi-1002082-g001: Ribbon structures of the KD-AID fragment.(a) Comparison of the unphosphorylated KD-AID fragment (PDB Id: 3H4J, blue ribbons) that represents the open state with the homologous phosphorylated KD fragment (PDB Id: 3DAE, red ribbons) representing the closed state. (b) The N-terminal lobe (residues 27–114) and part of the C-terminal lobe (residues 115–175) of the KD domain are shown in orange, and are used to measure the interlobe conformational transition. The activation loop (residues 176–206) is shown in pink, with the rest of KD shown in green, and the AID fragment shown in red.
Mentions: Biochemical experiments have shown that the isolated full-length α-subunit and even the α1 isoform (residues 1–392) have little activity due to the presence of the conserved AID domain [10], [11]. Recently, an exciting X-ray crystallography study [12] has successfully crystallized an unphosphorylated fragment containing both KD and AID from Schizosaccharomyces pombe (PDB Id: 3H4J), and a phosphorylated fragment containing only KD from Saccharomyces cerevisiae (PDB Id: 3DAE), providing a static structural view of how AID inhibits the conformational transition of the N-terminal and C-terminal lobes in the KD domain to the functional closed state (Figure 1a) [13]. Mutagenesis of key residues of AID were found to restore catalytic function of the KD fragment, thereby isolating key residue interactions between these two domains. The same interface point mutations of the rat AMPK α1 subunit show exactly the same catalytic trends as the S.Pombe KD-AID fragment, which was further confirmed in the rat AMPK holoenzyme in that these same mutations both increase the catalytic activity and slow the dephosphorylation of the α-subunit, independent of AMP concentration. Based on the X-ray crystal structures and catalytic activity upon mutagenesis, the authors proposed a new conformational switch model for the regulatory mechanism of AMPK activity in which the interaction of AID with KD requires the latter to adopt a relatively open conformational form that is inactive. The eventual binding of AMP to the γ-subunit changes the interactions between the AID and KD, at present by an unknown molecular mechanism, to remove the inhibitory effect of AID to allow the interlobe conformational transition to the closed state.

Bottom Line: However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited.By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition.Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop.

View Article: PubMed Central - PubMed

Affiliation: MOE-Microsoft Key Laboratory for Intelligent Computing and Intelligent Systems, Department of Computer Science and Engineering, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
We use a novel normal mode analysis of an elastic network model drawn from configurations generated during microsecond all-atom molecular dynamics simulations to analyze the mechanism of auto-inhibition of AMP-activated protein kinase (AMPK). A recent X-ray and mutagenesis experiment (Chen, et al Nature 2009, 459, 1146) of the AMPK homolog S. Pombe sucrose non-fermenting 1 (SNF1) has proposed a new conformational switch model involving the movement of the kinase domain (KD) between an inactive unphosphorylated open state and an active or semi-active phosphorylated closed state, mediated by the autoinhibitory domain (AID), and a similar mutagenesis study showed that rat AMPK has the same auto-inhibition mechanism. However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited. By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition. Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop.

Show MeSH