A conserved cis-proline precludes metal binding by the active site thiolates in members of the thioredoxin family of proteins

Dan Su, Carsten Berndt, Dmitri Fomenko, Arne Holmgren, Vadim N. Gladyshev

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Many thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S 2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site.

Original languageEnglish (US)
Pages (from-to)6903-6910
Number of pages8
JournalBiochemistry
Volume46
Issue number23
DOIs
StatePublished - Jun 12 2007

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Thioredoxins
Proline
Catalytic Domain
Metals
Binding Sites
Proteins
Peroxiredoxins
Cysteine
Mutation
Copper
Protein Disulfide Reductase (Glutathione)
Mutagenesis
Trace Elements
Threonine
Histidine
Alanine
Serine
Functional groups
Arginine

ASJC Scopus subject areas

  • Biochemistry

Cite this

A conserved cis-proline precludes metal binding by the active site thiolates in members of the thioredoxin family of proteins. / Su, Dan; Berndt, Carsten; Fomenko, Dmitri; Holmgren, Arne; Gladyshev, Vadim N.

In: Biochemistry, Vol. 46, No. 23, 12.06.2007, p. 6903-6910.

Research output: Contribution to journalArticle

Su, Dan ; Berndt, Carsten ; Fomenko, Dmitri ; Holmgren, Arne ; Gladyshev, Vadim N. / A conserved cis-proline precludes metal binding by the active site thiolates in members of the thioredoxin family of proteins. In: Biochemistry. 2007 ; Vol. 46, No. 23. pp. 6903-6910.
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abstract = "Many thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S 2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site.",
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