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Loop interactions and dynamics tune the enzymatic activity of the human histone deacetylase 8.

Kunze MB, Wright DW, Werbeck ND, Kirkpatrick J, Coveney PV, Hansen DF - J. Am. Chem. Soc. (2013)

Bottom Line: Yet it has remained unclear how the dynamics of the entrance surface tune and influence the catalytic activity of HDAC8.Using long time scale all atom molecular dynamics simulations we have found a mechanism whereby the interactions and dynamics of two loops tune the configuration of functionally important residues of HDAC8 and could therefore influence the activity of the enzyme.Our work delivers detailed insight into the dynamic loop network of HDAC8 and provides an explanation for a number of experimental observations.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London , Gower Street, London WC1E 6BT, United Kingdom.

ABSTRACT
The human histone deacetylase 8 (HDAC8) is a key hydrolase in gene regulation and has been identified as a drug target for the treatment of several cancers. Previously the HDAC8 enzyme has been extensively studied using biochemical techniques, X-ray crystallography, and computational methods. Those investigations have yielded detailed information about the active site and have demonstrated that the substrate entrance surface is highly dynamic. Yet it has remained unclear how the dynamics of the entrance surface tune and influence the catalytic activity of HDAC8. Using long time scale all atom molecular dynamics simulations we have found a mechanism whereby the interactions and dynamics of two loops tune the configuration of functionally important residues of HDAC8 and could therefore influence the activity of the enzyme. We subsequently investigated this hypothesis using a well-established fluorescence activity assay and a noninvasive real-time progression assay, where deacetylation of a p53 based peptide was observed by nuclear magnetic resonance spectroscopy. Our work delivers detailed insight into the dynamic loop network of HDAC8 and provides an explanation for a number of experimental observations.

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(a) Ribbonrepresentation of snapshots during the simulation showingthe ‘in’ (pale colors) and ‘out’ (darkcolors) conformation of the binding rail (green residues). Microkineticprocesses including their localization are annotated. (b) Microkineticprocesses and states over the simulation time: (i) binding rail flips,measured via Φ of Tyr100 (ii) L1:L2 salt-bridge formation betweenLys33 and Asp87–89 measured using a Lys Nζ-Asp Oδ distance cutoff of 0.35 nm (iii) presenceof an α-helix at residues 93–97 as calculated by STRIDE.
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fig2: (a) Ribbonrepresentation of snapshots during the simulation showingthe ‘in’ (pale colors) and ‘out’ (darkcolors) conformation of the binding rail (green residues). Microkineticprocesses including their localization are annotated. (b) Microkineticprocesses and states over the simulation time: (i) binding rail flips,measured via Φ of Tyr100 (ii) L1:L2 salt-bridge formation betweenLys33 and Asp87–89 measured using a Lys Nζ-Asp Oδ distance cutoff of 0.35 nm (iii) presenceof an α-helix at residues 93–97 as calculated by STRIDE.

Mentions: As expected from the previously solved structuresand experimental results, the simulation of free HDAC8 shows thatthe surface in the vicinity of the entrance to the catalytic siteis very flexible and in particular the L1 and L2 loops of HDAC8 interconvertbetween different states (Figure 2), some ofwhich are closely related to different crystal structures (Figure S1). For example, in one of the L1 loopconformations present during the simulation an extra cavity and alarge groove become accessible, as has been observed in crystal structureswith different ligands10,12 and in other computational studies.14,15 For the L2 loop, the binding rail has two distinct conformations,which we will refer to as ‘in’ and ‘out’(colored light- and dark-green, respectively, in Figure 2a). In the ‘in’ conformation Tyr100 and Asp101build a rail toward the catalytic site as observed in crystal structures10,13 (Figures 1b and S1d andVideo S1), which has been attributed to substrate binding andpositioning (Figure 1b).10 On the contrary, in the ‘out’ conformation,the binding rail residues 100 and 101 are oriented away from the entranceto the catalytic site. The interconversion between ‘in’and ‘out’ conformations takes place at Tyr100, as shownin the analysis of the backbone Φ angle (Figure 2b). The Φ angle is larger than −60° in the‘in’ conformation, as illustrated in Figure S1d, where, for example, the binding rail is mainlyin the ‘in’ conformation between 800 and 1000 ns.


Loop interactions and dynamics tune the enzymatic activity of the human histone deacetylase 8.

Kunze MB, Wright DW, Werbeck ND, Kirkpatrick J, Coveney PV, Hansen DF - J. Am. Chem. Soc. (2013)

(a) Ribbonrepresentation of snapshots during the simulation showingthe ‘in’ (pale colors) and ‘out’ (darkcolors) conformation of the binding rail (green residues). Microkineticprocesses including their localization are annotated. (b) Microkineticprocesses and states over the simulation time: (i) binding rail flips,measured via Φ of Tyr100 (ii) L1:L2 salt-bridge formation betweenLys33 and Asp87–89 measured using a Lys Nζ-Asp Oδ distance cutoff of 0.35 nm (iii) presenceof an α-helix at residues 93–97 as calculated by STRIDE.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926704&req=5

fig2: (a) Ribbonrepresentation of snapshots during the simulation showingthe ‘in’ (pale colors) and ‘out’ (darkcolors) conformation of the binding rail (green residues). Microkineticprocesses including their localization are annotated. (b) Microkineticprocesses and states over the simulation time: (i) binding rail flips,measured via Φ of Tyr100 (ii) L1:L2 salt-bridge formation betweenLys33 and Asp87–89 measured using a Lys Nζ-Asp Oδ distance cutoff of 0.35 nm (iii) presenceof an α-helix at residues 93–97 as calculated by STRIDE.
Mentions: As expected from the previously solved structuresand experimental results, the simulation of free HDAC8 shows thatthe surface in the vicinity of the entrance to the catalytic siteis very flexible and in particular the L1 and L2 loops of HDAC8 interconvertbetween different states (Figure 2), some ofwhich are closely related to different crystal structures (Figure S1). For example, in one of the L1 loopconformations present during the simulation an extra cavity and alarge groove become accessible, as has been observed in crystal structureswith different ligands10,12 and in other computational studies.14,15 For the L2 loop, the binding rail has two distinct conformations,which we will refer to as ‘in’ and ‘out’(colored light- and dark-green, respectively, in Figure 2a). In the ‘in’ conformation Tyr100 and Asp101build a rail toward the catalytic site as observed in crystal structures10,13 (Figures 1b and S1d andVideo S1), which has been attributed to substrate binding andpositioning (Figure 1b).10 On the contrary, in the ‘out’ conformation,the binding rail residues 100 and 101 are oriented away from the entranceto the catalytic site. The interconversion between ‘in’and ‘out’ conformations takes place at Tyr100, as shownin the analysis of the backbone Φ angle (Figure 2b). The Φ angle is larger than −60° in the‘in’ conformation, as illustrated in Figure S1d, where, for example, the binding rail is mainlyin the ‘in’ conformation between 800 and 1000 ns.

Bottom Line: Yet it has remained unclear how the dynamics of the entrance surface tune and influence the catalytic activity of HDAC8.Using long time scale all atom molecular dynamics simulations we have found a mechanism whereby the interactions and dynamics of two loops tune the configuration of functionally important residues of HDAC8 and could therefore influence the activity of the enzyme.Our work delivers detailed insight into the dynamic loop network of HDAC8 and provides an explanation for a number of experimental observations.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London , Gower Street, London WC1E 6BT, United Kingdom.

ABSTRACT
The human histone deacetylase 8 (HDAC8) is a key hydrolase in gene regulation and has been identified as a drug target for the treatment of several cancers. Previously the HDAC8 enzyme has been extensively studied using biochemical techniques, X-ray crystallography, and computational methods. Those investigations have yielded detailed information about the active site and have demonstrated that the substrate entrance surface is highly dynamic. Yet it has remained unclear how the dynamics of the entrance surface tune and influence the catalytic activity of HDAC8. Using long time scale all atom molecular dynamics simulations we have found a mechanism whereby the interactions and dynamics of two loops tune the configuration of functionally important residues of HDAC8 and could therefore influence the activity of the enzyme. We subsequently investigated this hypothesis using a well-established fluorescence activity assay and a noninvasive real-time progression assay, where deacetylation of a p53 based peptide was observed by nuclear magnetic resonance spectroscopy. Our work delivers detailed insight into the dynamic loop network of HDAC8 and provides an explanation for a number of experimental observations.

Show MeSH
Related in: MedlinePlus