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Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening.

Maier EM, Gersting SW, Kemter KF, Jank JM, Reindl M, Messing DD, Truger MS, Sommerhoff CP, Muntau AC - Hum. Mol. Genet. (2009)

Bottom Line: This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants.Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity.Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pediatrics, Children's Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

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Related in: MedlinePlus

Functional and conformational involvement of amino acid residues affected by MCAD mutations in networks of side-chain interactions. Selected parts of subunit backbones are shown as ribbon representations. Selected residues are depicted as stick models with carbon atoms in white, oxygen atoms in red and nitrogen atoms in blue. Hydrogen bonds and electrostatic interactions are shown as golden dotted lines. The cofactor FAD and the substrate analog 3-thiaoctanoyl-CoA are shown in yellow and in purple, respectively. (A) Residue Y133 is located at the beginning of the β-domain of the protein (subunit A, blue). It is part of the active site crevice by binding the isoalloxazine ring of FAD, together with T136 and T168. The aromatic side chain of Y133 establishes hydrophobic interactions with A100, L103, P107, V135, F177, A248, F252 and V259. These residues shape the hydrophobic core of the deep binding cavity for the fatty acid portion of the substrate. The precise three-dimensional arrangement of FAD, the Cα–Cβ bond of the substrate and the catalytic base E376 is the basis for enzyme function. Moreover, the structural integrity of the hydrophobic core of this cavity is crucial for the assembly of tetramers from dimers by four-helix bundle interactions between α-helices G and H in one subunit (subunit A, blue) and α helices I and K in the opposite unit of the adjacent dimer (subunit D, red). (B) The guanidinium side-chain of R181 adopts a central position in the extended loop structure (residues 182–193) at the surface of the protein that connects β-strands 4 and 5 (subunit A, blue). R181 networks with loop residues D183, D185, K187 and A188 via hydrogen bonds, and this contributes to the correct spatial conformation of the loop. The neighboring S182 interacts with T195 in β-strand 5, which establishes interactions to the loop (residues 239–244) interconnecting the β-domain with the C-terminal α-domain. The following α-helix G is part of both the hydrophobic core of the active site and the four-helix bundle between α-helices G and H in one subunit (subunit A, blue) and α-helices I and K in the opposite unit of the adjacent dimer (subunit D, red) assembling the tetramer. The active site is represented by the cofactor FAD interacting with Y133, T136 and T168, the Cα–Cβ bond of the substrate, and the catalytic base E376. (C) Residue R388 is located in the C-terminal α-helix K and is part of an interface between two subunits forming the dimer (subunit A, blue; subunit B, yellow). The interface defines a funnel-shaped crevice, the entrance to the active site. Subunit A (blue) lines the crevice with α-helix K and the loops between β-strands 4 and 5 (containing R181) and α-helices H and I. The adjacent subunit B (yellow) contributes to the interface via the loop between α-helices G and H. R388 joins a network of hydrogen bonds and electrostatic interactions comprising residues E389, R324, N325, D253 and S191, which bind the CoA moiety of the substrate to the entrance of the active site (subunit A, blue). R281 and T283 in the adjacent subunit (subunit B, yellow) interact with the pyrophosphate and the adenine ring of the cofactor FAD.
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DDP079F5: Functional and conformational involvement of amino acid residues affected by MCAD mutations in networks of side-chain interactions. Selected parts of subunit backbones are shown as ribbon representations. Selected residues are depicted as stick models with carbon atoms in white, oxygen atoms in red and nitrogen atoms in blue. Hydrogen bonds and electrostatic interactions are shown as golden dotted lines. The cofactor FAD and the substrate analog 3-thiaoctanoyl-CoA are shown in yellow and in purple, respectively. (A) Residue Y133 is located at the beginning of the β-domain of the protein (subunit A, blue). It is part of the active site crevice by binding the isoalloxazine ring of FAD, together with T136 and T168. The aromatic side chain of Y133 establishes hydrophobic interactions with A100, L103, P107, V135, F177, A248, F252 and V259. These residues shape the hydrophobic core of the deep binding cavity for the fatty acid portion of the substrate. The precise three-dimensional arrangement of FAD, the Cα–Cβ bond of the substrate and the catalytic base E376 is the basis for enzyme function. Moreover, the structural integrity of the hydrophobic core of this cavity is crucial for the assembly of tetramers from dimers by four-helix bundle interactions between α-helices G and H in one subunit (subunit A, blue) and α helices I and K in the opposite unit of the adjacent dimer (subunit D, red). (B) The guanidinium side-chain of R181 adopts a central position in the extended loop structure (residues 182–193) at the surface of the protein that connects β-strands 4 and 5 (subunit A, blue). R181 networks with loop residues D183, D185, K187 and A188 via hydrogen bonds, and this contributes to the correct spatial conformation of the loop. The neighboring S182 interacts with T195 in β-strand 5, which establishes interactions to the loop (residues 239–244) interconnecting the β-domain with the C-terminal α-domain. The following α-helix G is part of both the hydrophobic core of the active site and the four-helix bundle between α-helices G and H in one subunit (subunit A, blue) and α-helices I and K in the opposite unit of the adjacent dimer (subunit D, red) assembling the tetramer. The active site is represented by the cofactor FAD interacting with Y133, T136 and T168, the Cα–Cβ bond of the substrate, and the catalytic base E376. (C) Residue R388 is located in the C-terminal α-helix K and is part of an interface between two subunits forming the dimer (subunit A, blue; subunit B, yellow). The interface defines a funnel-shaped crevice, the entrance to the active site. Subunit A (blue) lines the crevice with α-helix K and the loops between β-strands 4 and 5 (containing R181) and α-helices H and I. The adjacent subunit B (yellow) contributes to the interface via the loop between α-helices G and H. R388 joins a network of hydrogen bonds and electrostatic interactions comprising residues E389, R324, N325, D253 and S191, which bind the CoA moiety of the substrate to the entrance of the active site (subunit A, blue). R281 and T283 in the adjacent subunit (subunit B, yellow) interact with the pyrophosphate and the adenine ring of the cofactor FAD.

Mentions: Residue Y133 maps to the β-sheet domain (residues 130–239) and is an essential part of the active site (Fig. 5A). It directly interacts with the cofactor FAD via hydrogen bond formation. Its aromatic side-chain points towards the hydrophobic core of the deep binding cavity for the fatty acid portion of the substrate establishing hydrophobic interactions with the residues L103, V135, F177, A248 and F252 which line the cavity. To poise the Cα–Cβ bond of the fatty acid for dehydrogenation, it is sandwiched between the re-face of the isoalloxazine moiety of FAD and the catalytic base E376. A replacement of the large, hydrophobic tyrosine by the smaller, positively charged histidine is supposed to distort the hydrophobic packing of the binding cavity and by this to lead to a conformational rearrangement of the active site pocket. As a result, the correct 3D arrangement of FAD, the Cα–Cβ bond of the substrate and the catalytic base E376 will be impaired leading to the marked disturbance in enzyme function. Moreover, A248 and F252 are located within the α-helix G. Interactions between α-helices G and H in one subunit of the dimer and α-helices I and K in the opposite unit of the adjacent dimer are known to promote MCAD tetramer assembly from dimers. Hence, conformational alteration of the substrate binding cavity with impairment of the helix–helix interactions due to the Y133H substitution might explain the observed distortion of the oligomeric state.


Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening.

Maier EM, Gersting SW, Kemter KF, Jank JM, Reindl M, Messing DD, Truger MS, Sommerhoff CP, Muntau AC - Hum. Mol. Genet. (2009)

Functional and conformational involvement of amino acid residues affected by MCAD mutations in networks of side-chain interactions. Selected parts of subunit backbones are shown as ribbon representations. Selected residues are depicted as stick models with carbon atoms in white, oxygen atoms in red and nitrogen atoms in blue. Hydrogen bonds and electrostatic interactions are shown as golden dotted lines. The cofactor FAD and the substrate analog 3-thiaoctanoyl-CoA are shown in yellow and in purple, respectively. (A) Residue Y133 is located at the beginning of the β-domain of the protein (subunit A, blue). It is part of the active site crevice by binding the isoalloxazine ring of FAD, together with T136 and T168. The aromatic side chain of Y133 establishes hydrophobic interactions with A100, L103, P107, V135, F177, A248, F252 and V259. These residues shape the hydrophobic core of the deep binding cavity for the fatty acid portion of the substrate. The precise three-dimensional arrangement of FAD, the Cα–Cβ bond of the substrate and the catalytic base E376 is the basis for enzyme function. Moreover, the structural integrity of the hydrophobic core of this cavity is crucial for the assembly of tetramers from dimers by four-helix bundle interactions between α-helices G and H in one subunit (subunit A, blue) and α helices I and K in the opposite unit of the adjacent dimer (subunit D, red). (B) The guanidinium side-chain of R181 adopts a central position in the extended loop structure (residues 182–193) at the surface of the protein that connects β-strands 4 and 5 (subunit A, blue). R181 networks with loop residues D183, D185, K187 and A188 via hydrogen bonds, and this contributes to the correct spatial conformation of the loop. The neighboring S182 interacts with T195 in β-strand 5, which establishes interactions to the loop (residues 239–244) interconnecting the β-domain with the C-terminal α-domain. The following α-helix G is part of both the hydrophobic core of the active site and the four-helix bundle between α-helices G and H in one subunit (subunit A, blue) and α-helices I and K in the opposite unit of the adjacent dimer (subunit D, red) assembling the tetramer. The active site is represented by the cofactor FAD interacting with Y133, T136 and T168, the Cα–Cβ bond of the substrate, and the catalytic base E376. (C) Residue R388 is located in the C-terminal α-helix K and is part of an interface between two subunits forming the dimer (subunit A, blue; subunit B, yellow). The interface defines a funnel-shaped crevice, the entrance to the active site. Subunit A (blue) lines the crevice with α-helix K and the loops between β-strands 4 and 5 (containing R181) and α-helices H and I. The adjacent subunit B (yellow) contributes to the interface via the loop between α-helices G and H. R388 joins a network of hydrogen bonds and electrostatic interactions comprising residues E389, R324, N325, D253 and S191, which bind the CoA moiety of the substrate to the entrance of the active site (subunit A, blue). R281 and T283 in the adjacent subunit (subunit B, yellow) interact with the pyrophosphate and the adenine ring of the cofactor FAD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667288&req=5

DDP079F5: Functional and conformational involvement of amino acid residues affected by MCAD mutations in networks of side-chain interactions. Selected parts of subunit backbones are shown as ribbon representations. Selected residues are depicted as stick models with carbon atoms in white, oxygen atoms in red and nitrogen atoms in blue. Hydrogen bonds and electrostatic interactions are shown as golden dotted lines. The cofactor FAD and the substrate analog 3-thiaoctanoyl-CoA are shown in yellow and in purple, respectively. (A) Residue Y133 is located at the beginning of the β-domain of the protein (subunit A, blue). It is part of the active site crevice by binding the isoalloxazine ring of FAD, together with T136 and T168. The aromatic side chain of Y133 establishes hydrophobic interactions with A100, L103, P107, V135, F177, A248, F252 and V259. These residues shape the hydrophobic core of the deep binding cavity for the fatty acid portion of the substrate. The precise three-dimensional arrangement of FAD, the Cα–Cβ bond of the substrate and the catalytic base E376 is the basis for enzyme function. Moreover, the structural integrity of the hydrophobic core of this cavity is crucial for the assembly of tetramers from dimers by four-helix bundle interactions between α-helices G and H in one subunit (subunit A, blue) and α helices I and K in the opposite unit of the adjacent dimer (subunit D, red). (B) The guanidinium side-chain of R181 adopts a central position in the extended loop structure (residues 182–193) at the surface of the protein that connects β-strands 4 and 5 (subunit A, blue). R181 networks with loop residues D183, D185, K187 and A188 via hydrogen bonds, and this contributes to the correct spatial conformation of the loop. The neighboring S182 interacts with T195 in β-strand 5, which establishes interactions to the loop (residues 239–244) interconnecting the β-domain with the C-terminal α-domain. The following α-helix G is part of both the hydrophobic core of the active site and the four-helix bundle between α-helices G and H in one subunit (subunit A, blue) and α-helices I and K in the opposite unit of the adjacent dimer (subunit D, red) assembling the tetramer. The active site is represented by the cofactor FAD interacting with Y133, T136 and T168, the Cα–Cβ bond of the substrate, and the catalytic base E376. (C) Residue R388 is located in the C-terminal α-helix K and is part of an interface between two subunits forming the dimer (subunit A, blue; subunit B, yellow). The interface defines a funnel-shaped crevice, the entrance to the active site. Subunit A (blue) lines the crevice with α-helix K and the loops between β-strands 4 and 5 (containing R181) and α-helices H and I. The adjacent subunit B (yellow) contributes to the interface via the loop between α-helices G and H. R388 joins a network of hydrogen bonds and electrostatic interactions comprising residues E389, R324, N325, D253 and S191, which bind the CoA moiety of the substrate to the entrance of the active site (subunit A, blue). R281 and T283 in the adjacent subunit (subunit B, yellow) interact with the pyrophosphate and the adenine ring of the cofactor FAD.
Mentions: Residue Y133 maps to the β-sheet domain (residues 130–239) and is an essential part of the active site (Fig. 5A). It directly interacts with the cofactor FAD via hydrogen bond formation. Its aromatic side-chain points towards the hydrophobic core of the deep binding cavity for the fatty acid portion of the substrate establishing hydrophobic interactions with the residues L103, V135, F177, A248 and F252 which line the cavity. To poise the Cα–Cβ bond of the fatty acid for dehydrogenation, it is sandwiched between the re-face of the isoalloxazine moiety of FAD and the catalytic base E376. A replacement of the large, hydrophobic tyrosine by the smaller, positively charged histidine is supposed to distort the hydrophobic packing of the binding cavity and by this to lead to a conformational rearrangement of the active site pocket. As a result, the correct 3D arrangement of FAD, the Cα–Cβ bond of the substrate and the catalytic base E376 will be impaired leading to the marked disturbance in enzyme function. Moreover, A248 and F252 are located within the α-helix G. Interactions between α-helices G and H in one subunit of the dimer and α-helices I and K in the opposite unit of the adjacent dimer are known to promote MCAD tetramer assembly from dimers. Hence, conformational alteration of the substrate binding cavity with impairment of the helix–helix interactions due to the Y133H substitution might explain the observed distortion of the oligomeric state.

Bottom Line: This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants.Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity.Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pediatrics, Children's Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

Show MeSH
Related in: MedlinePlus