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Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets.

Palermo G, Bauer I, Campomanes P, Cavalli A, Armirotti A, Girotto S, Rothlisberger U, De Vivo M - PLoS Comput. Biol. (2015)

Bottom Line: Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH.Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture.The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Genova, Italy.

ABSTRACT
The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

No MeSH data available.


Related in: MedlinePlus

Polar plots of the φ angle (dihedral angle along the Cα-Cβ axis) of Phe432 (φF—green dots) and Trp531 (φW—violet dots) with respect to the d-MA (red background), d-T (cyan background), and d-AB (yellow background) distances for pre-reactive conformations of the wtFAAH/anandamide (first row), wtFAAH/oleamide (second row), and wtFAAH/palmitoylethanolamide (PEA—third row) systems.The polar (d-MA, d-T and d-AB) and angular (φ—in red on the plot) coordinates are explicitly indicated on the plots. The approximate values of φF of Phe432 for the “open” and “closed” MA channel configurations are highlighted with blue dashed bars. Distances and angles are expressed in Å and degrees, respectively. Definitions of the d-MA, d-T and d-AB distances are reported in the Methods section. Selected snapshots from MD simulations indicating the “open” (left) and “closed” (right) MA channel configurations, as induced by the rotation of the φ angle of Phe432 and the cooperative Trp531, are shown at the bottom of the polar plots. The MA (red) and AB (orange) channels are represented in molecular surfaces. Phe432 (green) and Trp531 (violet) are shown in space-filling representation. The φ angle of the two residues is explicitly reported.
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pcbi.1004231.g005: Polar plots of the φ angle (dihedral angle along the Cα-Cβ axis) of Phe432 (φF—green dots) and Trp531 (φW—violet dots) with respect to the d-MA (red background), d-T (cyan background), and d-AB (yellow background) distances for pre-reactive conformations of the wtFAAH/anandamide (first row), wtFAAH/oleamide (second row), and wtFAAH/palmitoylethanolamide (PEA—third row) systems.The polar (d-MA, d-T and d-AB) and angular (φ—in red on the plot) coordinates are explicitly indicated on the plots. The approximate values of φF of Phe432 for the “open” and “closed” MA channel configurations are highlighted with blue dashed bars. Distances and angles are expressed in Å and degrees, respectively. Definitions of the d-MA, d-T and d-AB distances are reported in the Methods section. Selected snapshots from MD simulations indicating the “open” (left) and “closed” (right) MA channel configurations, as induced by the rotation of the φ angle of Phe432 and the cooperative Trp531, are shown at the bottom of the polar plots. The MA (red) and AB (orange) channels are represented in molecular surfaces. Phe432 (green) and Trp531 (violet) are shown in space-filling representation. The φ angle of the two residues is explicitly reported.

Mentions: Phe432 and Trp531 trigger the MA<–>AB transitions of anandamide, assuming different configurations that open and close the MA channel (Fig 2). This mechanism favors the proper location of pre-reactive conformations of anandamide between the two channels, as evidenced by the polar plot of the φ angles of Phe432 (φF) and Trp531 (φW) with respect to the location of the substrate in pre-reactive states (Fig 5). In detail, for pre-reactive conformations in the MA channel (red plot), the φF (green dots) ranges from ~120° to ~180° with the opening of MA. During the MA<–>AB transfer (cyan plot), the φF shows a bimodal distribution, given the rotation of φF from ~150° (“open” MA channel) to ~60° (“closed” MA channel, as observed in the X-ray structure), which permits the MA<–>AB transfer of the arachidonoyl chain. Trp531 contributes to this transfer, rotating φW by about ~35/40° (magenta dots). When pre-reactive conformations are in AB (yellow plot), Phe432 mainly closes the MA channel (φF ~65°), opening the adjacent AB channel, while Trp531 rotates φW from ~145° to ~180°.


Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets.

Palermo G, Bauer I, Campomanes P, Cavalli A, Armirotti A, Girotto S, Rothlisberger U, De Vivo M - PLoS Comput. Biol. (2015)

Polar plots of the φ angle (dihedral angle along the Cα-Cβ axis) of Phe432 (φF—green dots) and Trp531 (φW—violet dots) with respect to the d-MA (red background), d-T (cyan background), and d-AB (yellow background) distances for pre-reactive conformations of the wtFAAH/anandamide (first row), wtFAAH/oleamide (second row), and wtFAAH/palmitoylethanolamide (PEA—third row) systems.The polar (d-MA, d-T and d-AB) and angular (φ—in red on the plot) coordinates are explicitly indicated on the plots. The approximate values of φF of Phe432 for the “open” and “closed” MA channel configurations are highlighted with blue dashed bars. Distances and angles are expressed in Å and degrees, respectively. Definitions of the d-MA, d-T and d-AB distances are reported in the Methods section. Selected snapshots from MD simulations indicating the “open” (left) and “closed” (right) MA channel configurations, as induced by the rotation of the φ angle of Phe432 and the cooperative Trp531, are shown at the bottom of the polar plots. The MA (red) and AB (orange) channels are represented in molecular surfaces. Phe432 (green) and Trp531 (violet) are shown in space-filling representation. The φ angle of the two residues is explicitly reported.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481349&req=5

pcbi.1004231.g005: Polar plots of the φ angle (dihedral angle along the Cα-Cβ axis) of Phe432 (φF—green dots) and Trp531 (φW—violet dots) with respect to the d-MA (red background), d-T (cyan background), and d-AB (yellow background) distances for pre-reactive conformations of the wtFAAH/anandamide (first row), wtFAAH/oleamide (second row), and wtFAAH/palmitoylethanolamide (PEA—third row) systems.The polar (d-MA, d-T and d-AB) and angular (φ—in red on the plot) coordinates are explicitly indicated on the plots. The approximate values of φF of Phe432 for the “open” and “closed” MA channel configurations are highlighted with blue dashed bars. Distances and angles are expressed in Å and degrees, respectively. Definitions of the d-MA, d-T and d-AB distances are reported in the Methods section. Selected snapshots from MD simulations indicating the “open” (left) and “closed” (right) MA channel configurations, as induced by the rotation of the φ angle of Phe432 and the cooperative Trp531, are shown at the bottom of the polar plots. The MA (red) and AB (orange) channels are represented in molecular surfaces. Phe432 (green) and Trp531 (violet) are shown in space-filling representation. The φ angle of the two residues is explicitly reported.
Mentions: Phe432 and Trp531 trigger the MA<–>AB transitions of anandamide, assuming different configurations that open and close the MA channel (Fig 2). This mechanism favors the proper location of pre-reactive conformations of anandamide between the two channels, as evidenced by the polar plot of the φ angles of Phe432 (φF) and Trp531 (φW) with respect to the location of the substrate in pre-reactive states (Fig 5). In detail, for pre-reactive conformations in the MA channel (red plot), the φF (green dots) ranges from ~120° to ~180° with the opening of MA. During the MA<–>AB transfer (cyan plot), the φF shows a bimodal distribution, given the rotation of φF from ~150° (“open” MA channel) to ~60° (“closed” MA channel, as observed in the X-ray structure), which permits the MA<–>AB transfer of the arachidonoyl chain. Trp531 contributes to this transfer, rotating φW by about ~35/40° (magenta dots). When pre-reactive conformations are in AB (yellow plot), Phe432 mainly closes the MA channel (φF ~65°), opening the adjacent AB channel, while Trp531 rotates φW from ~145° to ~180°.

Bottom Line: Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH.Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture.The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Genova, Italy.

ABSTRACT
The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

No MeSH data available.


Related in: MedlinePlus