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A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model.

Min W, Angileri F, Luo H, Lauria A, Shanmugasundaram M, Almerico AM, Cappello F, de Macario EC, Lednev IK, Macario AJ, Robb FT - Sci Rep (2014)

Bottom Line: These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog.The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective.Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore; and Institute of Marine and Environmental Technology (IMET); Columbus Center, Baltimore, MD 21201, USA.

ABSTRACT
Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

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Related in: MedlinePlus

(A). Human CCT5 and P. furiosus PfCD superposed onto the crystal structure of Thermococcus strain KS-1 α. LEFT: The structure (Swiss-Model (http://swissmodel.expasy.org/) of PfCD (gold) superposed onto the crystal structure of KS1α (momomers, displayed in marine blue, violet, and deep teal as surface, and in cyan color as ribbon). The hexadecamer, double-ring structure, is depicted by a dotted line, as is on the right of the subsequent panels B and C. RIGHT: Magnified image of the superposed structures of the PfCD (gold) and Human CCT5 (orange) onto KS1α; cyan ribbon). Side chains of isoleucine at 138 of PfCD (blue) and isoleucine at 138 of KS1α (deep teal), and side chain of histidine at 147 of human CCT5 (red) are represented as ball and stick. AMP-PNP (green stick; also shown on the left in panels B and C) and magnesium ion (yellow ball). (B). PfCD around the Arg138 mutation and its predicted matching areas in the crystal structure of KS1α. LEFT: Mutant Arg138 (orange) and its adjacent Arg421 (red) in the opposite α helix are drawn with stick in the PfCD structure (marine blue). RIGHT: Arg138 mutation area was superposed onto the crystal structure of KS1α (monomers are displayed in various shades of blue). (C). Human CCT5 structure around the Arg147 mutation viewed from different angles and predicted matching areas in the crystal structure of KS1α. LEFT: Wild-type His147 (blue), mutant Arg147 (red) and their adjacent amino acids, Ser428 and Cys429 (magenta) in the opposite α helix are drawn with ball and stick and transparent surface in the human CCT5 structure (orange ribbon). RIGHT: The predicted Arg147 mutation area in the monomers is shown as a red surface on the crystal structure of KS1α (monomers are shown as surface in marine blue, violet, deep teal, and cyan colors.
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f2: (A). Human CCT5 and P. furiosus PfCD superposed onto the crystal structure of Thermococcus strain KS-1 α. LEFT: The structure (Swiss-Model (http://swissmodel.expasy.org/) of PfCD (gold) superposed onto the crystal structure of KS1α (momomers, displayed in marine blue, violet, and deep teal as surface, and in cyan color as ribbon). The hexadecamer, double-ring structure, is depicted by a dotted line, as is on the right of the subsequent panels B and C. RIGHT: Magnified image of the superposed structures of the PfCD (gold) and Human CCT5 (orange) onto KS1α; cyan ribbon). Side chains of isoleucine at 138 of PfCD (blue) and isoleucine at 138 of KS1α (deep teal), and side chain of histidine at 147 of human CCT5 (red) are represented as ball and stick. AMP-PNP (green stick; also shown on the left in panels B and C) and magnesium ion (yellow ball). (B). PfCD around the Arg138 mutation and its predicted matching areas in the crystal structure of KS1α. LEFT: Mutant Arg138 (orange) and its adjacent Arg421 (red) in the opposite α helix are drawn with stick in the PfCD structure (marine blue). RIGHT: Arg138 mutation area was superposed onto the crystal structure of KS1α (monomers are displayed in various shades of blue). (C). Human CCT5 structure around the Arg147 mutation viewed from different angles and predicted matching areas in the crystal structure of KS1α. LEFT: Wild-type His147 (blue), mutant Arg147 (red) and their adjacent amino acids, Ser428 and Cys429 (magenta) in the opposite α helix are drawn with ball and stick and transparent surface in the human CCT5 structure (orange ribbon). RIGHT: The predicted Arg147 mutation area in the monomers is shown as a red surface on the crystal structure of KS1α (monomers are shown as surface in marine blue, violet, deep teal, and cyan colors.

Mentions: Structural representations of the human CCT5 and PfCD were generated in the Swiss-Model server, using their primary amino-acid sequences and as template the crystal structure of the Cpn60 α subunit from Thermococcus KS-1 (KS-1α; PDB ID: 1Q3Q), which was crystallized with an ATP analogue, AMP-PNP, and fluoride ion in the active site23. Overall structural similarities and characteristic features of group II chaperonins are evident around the equatorial domain (Fig. 2A). This domain comprises the ATP/ADP binding site, is the region where the mutation H147R occurs in the case of the human CCT5-associated neuropathy, and is essentially superimposed. Noteworthy, the α-helixes in which are located His147 in CCT5, or Ile138 in PfCD overlap, and the side chain of His147 of human CCT5 and Ile138 of PfCD showed the same configuration as Ile138 of KS-1α. To analyze the structural consequences of mutation I138R in PfCD, we superimposed the PfCD structure onto the crystal structure of the KS-1α (1Q3Q) (Fig. 2B). The mutation I138R is located in the equatorial domain, in the N-terminus of Cpn, and this position is close to the intermediate domain, which serves as a hinge to allow binding of ATP/ADP when the apical alpha-helical protrusion domain of Cpn recognizes client proteins24. Arg138 is contiguous to the conserved motif Gly94-Thr99 constituting the ATP binding site in group II chaperonins25, suggesting that steric hindrance might impede the conformational change necessary for ATP binding. A similar situation is apparent in the case of mutant human CCT5 with Arg147, instead of His147 (Fig. 2C).


A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model.

Min W, Angileri F, Luo H, Lauria A, Shanmugasundaram M, Almerico AM, Cappello F, de Macario EC, Lednev IK, Macario AJ, Robb FT - Sci Rep (2014)

(A). Human CCT5 and P. furiosus PfCD superposed onto the crystal structure of Thermococcus strain KS-1 α. LEFT: The structure (Swiss-Model (http://swissmodel.expasy.org/) of PfCD (gold) superposed onto the crystal structure of KS1α (momomers, displayed in marine blue, violet, and deep teal as surface, and in cyan color as ribbon). The hexadecamer, double-ring structure, is depicted by a dotted line, as is on the right of the subsequent panels B and C. RIGHT: Magnified image of the superposed structures of the PfCD (gold) and Human CCT5 (orange) onto KS1α; cyan ribbon). Side chains of isoleucine at 138 of PfCD (blue) and isoleucine at 138 of KS1α (deep teal), and side chain of histidine at 147 of human CCT5 (red) are represented as ball and stick. AMP-PNP (green stick; also shown on the left in panels B and C) and magnesium ion (yellow ball). (B). PfCD around the Arg138 mutation and its predicted matching areas in the crystal structure of KS1α. LEFT: Mutant Arg138 (orange) and its adjacent Arg421 (red) in the opposite α helix are drawn with stick in the PfCD structure (marine blue). RIGHT: Arg138 mutation area was superposed onto the crystal structure of KS1α (monomers are displayed in various shades of blue). (C). Human CCT5 structure around the Arg147 mutation viewed from different angles and predicted matching areas in the crystal structure of KS1α. LEFT: Wild-type His147 (blue), mutant Arg147 (red) and their adjacent amino acids, Ser428 and Cys429 (magenta) in the opposite α helix are drawn with ball and stick and transparent surface in the human CCT5 structure (orange ribbon). RIGHT: The predicted Arg147 mutation area in the monomers is shown as a red surface on the crystal structure of KS1α (monomers are shown as surface in marine blue, violet, deep teal, and cyan colors.
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f2: (A). Human CCT5 and P. furiosus PfCD superposed onto the crystal structure of Thermococcus strain KS-1 α. LEFT: The structure (Swiss-Model (http://swissmodel.expasy.org/) of PfCD (gold) superposed onto the crystal structure of KS1α (momomers, displayed in marine blue, violet, and deep teal as surface, and in cyan color as ribbon). The hexadecamer, double-ring structure, is depicted by a dotted line, as is on the right of the subsequent panels B and C. RIGHT: Magnified image of the superposed structures of the PfCD (gold) and Human CCT5 (orange) onto KS1α; cyan ribbon). Side chains of isoleucine at 138 of PfCD (blue) and isoleucine at 138 of KS1α (deep teal), and side chain of histidine at 147 of human CCT5 (red) are represented as ball and stick. AMP-PNP (green stick; also shown on the left in panels B and C) and magnesium ion (yellow ball). (B). PfCD around the Arg138 mutation and its predicted matching areas in the crystal structure of KS1α. LEFT: Mutant Arg138 (orange) and its adjacent Arg421 (red) in the opposite α helix are drawn with stick in the PfCD structure (marine blue). RIGHT: Arg138 mutation area was superposed onto the crystal structure of KS1α (monomers are displayed in various shades of blue). (C). Human CCT5 structure around the Arg147 mutation viewed from different angles and predicted matching areas in the crystal structure of KS1α. LEFT: Wild-type His147 (blue), mutant Arg147 (red) and their adjacent amino acids, Ser428 and Cys429 (magenta) in the opposite α helix are drawn with ball and stick and transparent surface in the human CCT5 structure (orange ribbon). RIGHT: The predicted Arg147 mutation area in the monomers is shown as a red surface on the crystal structure of KS1α (monomers are shown as surface in marine blue, violet, deep teal, and cyan colors.
Mentions: Structural representations of the human CCT5 and PfCD were generated in the Swiss-Model server, using their primary amino-acid sequences and as template the crystal structure of the Cpn60 α subunit from Thermococcus KS-1 (KS-1α; PDB ID: 1Q3Q), which was crystallized with an ATP analogue, AMP-PNP, and fluoride ion in the active site23. Overall structural similarities and characteristic features of group II chaperonins are evident around the equatorial domain (Fig. 2A). This domain comprises the ATP/ADP binding site, is the region where the mutation H147R occurs in the case of the human CCT5-associated neuropathy, and is essentially superimposed. Noteworthy, the α-helixes in which are located His147 in CCT5, or Ile138 in PfCD overlap, and the side chain of His147 of human CCT5 and Ile138 of PfCD showed the same configuration as Ile138 of KS-1α. To analyze the structural consequences of mutation I138R in PfCD, we superimposed the PfCD structure onto the crystal structure of the KS-1α (1Q3Q) (Fig. 2B). The mutation I138R is located in the equatorial domain, in the N-terminus of Cpn, and this position is close to the intermediate domain, which serves as a hinge to allow binding of ATP/ADP when the apical alpha-helical protrusion domain of Cpn recognizes client proteins24. Arg138 is contiguous to the conserved motif Gly94-Thr99 constituting the ATP binding site in group II chaperonins25, suggesting that steric hindrance might impede the conformational change necessary for ATP binding. A similar situation is apparent in the case of mutant human CCT5 with Arg147, instead of His147 (Fig. 2C).

Bottom Line: These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog.The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective.Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore; and Institute of Marine and Environmental Technology (IMET); Columbus Center, Baltimore, MD 21201, USA.

ABSTRACT
Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

Show MeSH
Related in: MedlinePlus