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A gatekeeper helix determines the substrate specificity of Sjögren-Larsson Syndrome enzyme fatty aldehyde dehydrogenase.

Keller MA, Zander U, Fuchs JE, Kreutz C, Watschinger K, Mueller T, Golderer G, Liedl KR, Ralser M, Kräutler B, Werner ER, Marquez JA - Nat Commun (2014)

Bottom Line: Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase.The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases.Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria [2] Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis court Rd, Cambridge CB2 1GA, UK.

ABSTRACT
Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren-Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a 'gatekeeper' helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

No MeSH data available.


Related in: MedlinePlus

Membrane interaction of FALDH.(a) A patch of hydrophobic amino acids (shown in stick model) is located around the FALDH substrate funnel entrance, originating from loop V88-L92, helix G434-L438 (both in lightblue) and the gatekeeper helix 446–456 in green. Correct orientation of the gatekeeper helix is supported by coordination of K447 to the backbone of Y440 (distance: 2.7 Å). The transmembrane domain linker indicates the position of the C-terminal transmembrane membrane domain. (b) Sequence alignment of human and mouse class 3 aldehyde dehydrogenase isozymes for loop V88-L92, helix G434-L438 and the gatekeeper helix S446-L456 (with Clustal Omega, see Methods). Residue colour scheme: hydrophobic, red; polar, green; charged (neg.), blue; charged (pos.), violet. Secondary structure was predicted with the human FALDH structure and the Stride software (http://webclu.bio.wzw.tum.de/stride/). Grey areas indicate (when present) corresponding regions in different isozymes. The introduced K463X stop codon in the FALDH expression construct is highlighted with a star. Cysteine residues in ALDH3B1 (indicated by green areas) represent lipidation sites for subsequent membrane anchoring. (c) Comparison of the catalytic capacity (Vmax/KM) of FALDH with (K463X) and without (Q445X) the gatekeeper helix to metabolize octanal, dodecanal and hexadecanal. No difference was found for octadecanal. With hexadecanal, the Vmax/KM in Q445X was 10-fold lower (P=0.0003, t-test). Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (d) Proposed model for FALDH membrane interaction. FALDH dimer is anchored into lipid bilayers via two transmenbrane domains. Hydrophobic patches (including the gatekeeper helix) around the substrate funnel reach into the membrane and allow efficient substrate (shown in yellow) turnover in the active site (AS). Positively charged residues around these patches increase membrane interaction (see Supplementary Fig. 6a,b). NAD-binding pockets are directed away from the membrane and fully accessible.
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f5: Membrane interaction of FALDH.(a) A patch of hydrophobic amino acids (shown in stick model) is located around the FALDH substrate funnel entrance, originating from loop V88-L92, helix G434-L438 (both in lightblue) and the gatekeeper helix 446–456 in green. Correct orientation of the gatekeeper helix is supported by coordination of K447 to the backbone of Y440 (distance: 2.7 Å). The transmembrane domain linker indicates the position of the C-terminal transmembrane membrane domain. (b) Sequence alignment of human and mouse class 3 aldehyde dehydrogenase isozymes for loop V88-L92, helix G434-L438 and the gatekeeper helix S446-L456 (with Clustal Omega, see Methods). Residue colour scheme: hydrophobic, red; polar, green; charged (neg.), blue; charged (pos.), violet. Secondary structure was predicted with the human FALDH structure and the Stride software (http://webclu.bio.wzw.tum.de/stride/). Grey areas indicate (when present) corresponding regions in different isozymes. The introduced K463X stop codon in the FALDH expression construct is highlighted with a star. Cysteine residues in ALDH3B1 (indicated by green areas) represent lipidation sites for subsequent membrane anchoring. (c) Comparison of the catalytic capacity (Vmax/KM) of FALDH with (K463X) and without (Q445X) the gatekeeper helix to metabolize octanal, dodecanal and hexadecanal. No difference was found for octadecanal. With hexadecanal, the Vmax/KM in Q445X was 10-fold lower (P=0.0003, t-test). Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (d) Proposed model for FALDH membrane interaction. FALDH dimer is anchored into lipid bilayers via two transmenbrane domains. Hydrophobic patches (including the gatekeeper helix) around the substrate funnel reach into the membrane and allow efficient substrate (shown in yellow) turnover in the active site (AS). Positively charged residues around these patches increase membrane interaction (see Supplementary Fig. 6a,b). NAD-binding pockets are directed away from the membrane and fully accessible.

Mentions: A distinctive structural feature in human FALDH is the presence of a novel structural element represented by an alpha helix (S446-L456) that directly follows the dimerization domain. This short helical segment extends over the substrate entry tunnel and originates from the adjacent subunit in the FALDH dimer (Fig. 3 and Fig. 5d). A short linker segment at the C-terminal end (Fig. 2c), connects the helix to the predicted transmembrane region (not present in the structural model). The novel helix is formed by a series of well-conserved residues in the FALDH protein family, most of which are hydrophobic (Val-448, Trp-450, Gly-451, Phe-453, Phe-454, Leu-455 and Leu-456; Figs 3a,b, 5a and Supplementary Fig. 5). The main axis of this helix forms a 90° kink with the preceding helix formed by amino acids 433–439 located at the edge of the substrate tunnel rim. As a consequence, helix 446–456 crosses over the tunnel entry. Both polar and hydrophobic interactions stabilize the configuration of helix 446–456. For example, the terminal amine group of Leu-447 (from the helix itself) forms a hydrogen bonding network with backbone atoms in a preceding loop region formed by amino acids 440–443 (Fig. 5a). Conversely, the side chain of Leu-455 at its C-terminal end inserts into a hydrophobic patch formed by the side chains of Leu-438 and Leu-89 at opposite sides of the rim. In this configuration, the helical segment closes the access to the cavity and very likely prevents the passage of substrates inside or outside the cavity. At the same time, a series of hydrophobic residues in this helical segment, including Trp-450 and Phe-453 as well as Phe-454 in the C-terminal linker region and Phe-459, form a prominent hydrophobic region oriented towards the outside over the substrate site and directed towards the presumed positions of the membrane (Fig. 5a). Interestingly, Leu-455 and Leu-456 in the helix and linker regions, respectively, are oriented towards the inner side of the cavity, restricting the available space on the upper part of the tunnel. This induces a 93° kink in the direction of the substrate cavity as calculated using the MOLE toolkit for PyMOL42. This configuration suggested that this unique C-terminal region might have an important ‘gatekeeper’ function, by controlling the access to the catalytic site and restricting the available space in the upper region of the substrate cavity.


A gatekeeper helix determines the substrate specificity of Sjögren-Larsson Syndrome enzyme fatty aldehyde dehydrogenase.

Keller MA, Zander U, Fuchs JE, Kreutz C, Watschinger K, Mueller T, Golderer G, Liedl KR, Ralser M, Kräutler B, Werner ER, Marquez JA - Nat Commun (2014)

Membrane interaction of FALDH.(a) A patch of hydrophobic amino acids (shown in stick model) is located around the FALDH substrate funnel entrance, originating from loop V88-L92, helix G434-L438 (both in lightblue) and the gatekeeper helix 446–456 in green. Correct orientation of the gatekeeper helix is supported by coordination of K447 to the backbone of Y440 (distance: 2.7 Å). The transmembrane domain linker indicates the position of the C-terminal transmembrane membrane domain. (b) Sequence alignment of human and mouse class 3 aldehyde dehydrogenase isozymes for loop V88-L92, helix G434-L438 and the gatekeeper helix S446-L456 (with Clustal Omega, see Methods). Residue colour scheme: hydrophobic, red; polar, green; charged (neg.), blue; charged (pos.), violet. Secondary structure was predicted with the human FALDH structure and the Stride software (http://webclu.bio.wzw.tum.de/stride/). Grey areas indicate (when present) corresponding regions in different isozymes. The introduced K463X stop codon in the FALDH expression construct is highlighted with a star. Cysteine residues in ALDH3B1 (indicated by green areas) represent lipidation sites for subsequent membrane anchoring. (c) Comparison of the catalytic capacity (Vmax/KM) of FALDH with (K463X) and without (Q445X) the gatekeeper helix to metabolize octanal, dodecanal and hexadecanal. No difference was found for octadecanal. With hexadecanal, the Vmax/KM in Q445X was 10-fold lower (P=0.0003, t-test). Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (d) Proposed model for FALDH membrane interaction. FALDH dimer is anchored into lipid bilayers via two transmenbrane domains. Hydrophobic patches (including the gatekeeper helix) around the substrate funnel reach into the membrane and allow efficient substrate (shown in yellow) turnover in the active site (AS). Positively charged residues around these patches increase membrane interaction (see Supplementary Fig. 6a,b). NAD-binding pockets are directed away from the membrane and fully accessible.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109017&req=5

f5: Membrane interaction of FALDH.(a) A patch of hydrophobic amino acids (shown in stick model) is located around the FALDH substrate funnel entrance, originating from loop V88-L92, helix G434-L438 (both in lightblue) and the gatekeeper helix 446–456 in green. Correct orientation of the gatekeeper helix is supported by coordination of K447 to the backbone of Y440 (distance: 2.7 Å). The transmembrane domain linker indicates the position of the C-terminal transmembrane membrane domain. (b) Sequence alignment of human and mouse class 3 aldehyde dehydrogenase isozymes for loop V88-L92, helix G434-L438 and the gatekeeper helix S446-L456 (with Clustal Omega, see Methods). Residue colour scheme: hydrophobic, red; polar, green; charged (neg.), blue; charged (pos.), violet. Secondary structure was predicted with the human FALDH structure and the Stride software (http://webclu.bio.wzw.tum.de/stride/). Grey areas indicate (when present) corresponding regions in different isozymes. The introduced K463X stop codon in the FALDH expression construct is highlighted with a star. Cysteine residues in ALDH3B1 (indicated by green areas) represent lipidation sites for subsequent membrane anchoring. (c) Comparison of the catalytic capacity (Vmax/KM) of FALDH with (K463X) and without (Q445X) the gatekeeper helix to metabolize octanal, dodecanal and hexadecanal. No difference was found for octadecanal. With hexadecanal, the Vmax/KM in Q445X was 10-fold lower (P=0.0003, t-test). Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (d) Proposed model for FALDH membrane interaction. FALDH dimer is anchored into lipid bilayers via two transmenbrane domains. Hydrophobic patches (including the gatekeeper helix) around the substrate funnel reach into the membrane and allow efficient substrate (shown in yellow) turnover in the active site (AS). Positively charged residues around these patches increase membrane interaction (see Supplementary Fig. 6a,b). NAD-binding pockets are directed away from the membrane and fully accessible.
Mentions: A distinctive structural feature in human FALDH is the presence of a novel structural element represented by an alpha helix (S446-L456) that directly follows the dimerization domain. This short helical segment extends over the substrate entry tunnel and originates from the adjacent subunit in the FALDH dimer (Fig. 3 and Fig. 5d). A short linker segment at the C-terminal end (Fig. 2c), connects the helix to the predicted transmembrane region (not present in the structural model). The novel helix is formed by a series of well-conserved residues in the FALDH protein family, most of which are hydrophobic (Val-448, Trp-450, Gly-451, Phe-453, Phe-454, Leu-455 and Leu-456; Figs 3a,b, 5a and Supplementary Fig. 5). The main axis of this helix forms a 90° kink with the preceding helix formed by amino acids 433–439 located at the edge of the substrate tunnel rim. As a consequence, helix 446–456 crosses over the tunnel entry. Both polar and hydrophobic interactions stabilize the configuration of helix 446–456. For example, the terminal amine group of Leu-447 (from the helix itself) forms a hydrogen bonding network with backbone atoms in a preceding loop region formed by amino acids 440–443 (Fig. 5a). Conversely, the side chain of Leu-455 at its C-terminal end inserts into a hydrophobic patch formed by the side chains of Leu-438 and Leu-89 at opposite sides of the rim. In this configuration, the helical segment closes the access to the cavity and very likely prevents the passage of substrates inside or outside the cavity. At the same time, a series of hydrophobic residues in this helical segment, including Trp-450 and Phe-453 as well as Phe-454 in the C-terminal linker region and Phe-459, form a prominent hydrophobic region oriented towards the outside over the substrate site and directed towards the presumed positions of the membrane (Fig. 5a). Interestingly, Leu-455 and Leu-456 in the helix and linker regions, respectively, are oriented towards the inner side of the cavity, restricting the available space on the upper part of the tunnel. This induces a 93° kink in the direction of the substrate cavity as calculated using the MOLE toolkit for PyMOL42. This configuration suggested that this unique C-terminal region might have an important ‘gatekeeper’ function, by controlling the access to the catalytic site and restricting the available space in the upper region of the substrate cavity.

Bottom Line: Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase.The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases.Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria [2] Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis court Rd, Cambridge CB2 1GA, UK.

ABSTRACT
Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren-Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a 'gatekeeper' helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

No MeSH data available.


Related in: MedlinePlus