Human thioredoxin homodimers: Regulation by pH, role of aspartate 60, and crystal structure of the aspartate 60*[ asparagine mutant

John F. Andersen, David A R Sanders, John R. Gasdaska, Andrzej Weichsel, Garth Powis, William "Bill" Montfort

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Gys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (K(app)) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 μM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pK(a) of 6.5. A similar value was obtained using gel filtration at pH 3.8 (K(app) = 164 μM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by ~2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Ash 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for K(app) suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.

Original languageEnglish (US)
Pages (from-to)13979-13988
Number of pages10
JournalBiochemistry
Volume36
Issue number46
DOIs
StatePublished - Nov 18 1997

Fingerprint

Thioredoxins
Asparagine
Aspartic Acid
Dimers
Crystal structure
Diamide
Application programs
Disulfides
Gel Chromatography
Monomers
Ashes
Proteins
Gels
Oxidation-Reduction
Cell growth
Titration
Mutation
Water
Assays
Growth

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human thioredoxin homodimers : Regulation by pH, role of aspartate 60, and crystal structure of the aspartate 60*[ asparagine mutant. / Andersen, John F.; Sanders, David A R; Gasdaska, John R.; Weichsel, Andrzej; Powis, Garth; Montfort, William "Bill".

In: Biochemistry, Vol. 36, No. 46, 18.11.1997, p. 13979-13988.

Research output: Contribution to journalArticle

Andersen, John F. ; Sanders, David A R ; Gasdaska, John R. ; Weichsel, Andrzej ; Powis, Garth ; Montfort, William "Bill". / Human thioredoxin homodimers : Regulation by pH, role of aspartate 60, and crystal structure of the aspartate 60*[ asparagine mutant. In: Biochemistry. 1997 ; Vol. 36, No. 46. pp. 13979-13988.
@article{3259ba5cc90b4f9694de693046a05ea4,
title = "Human thioredoxin homodimers: Regulation by pH, role of aspartate 60, and crystal structure of the aspartate 60*[ asparagine mutant",
abstract = "Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Gys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (K(app)) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 μM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pK(a) of 6.5. A similar value was obtained using gel filtration at pH 3.8 (K(app) = 164 μM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by ~2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Ash 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for K(app) suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.",
author = "Andersen, {John F.} and Sanders, {David A R} and Gasdaska, {John R.} and Andrzej Weichsel and Garth Powis and Montfort, {William {"}Bill{"}}",
year = "1997",
month = "11",
day = "18",
doi = "10.1021/bi971004s",
language = "English (US)",
volume = "36",
pages = "13979--13988",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "46",

}

TY - JOUR

T1 - Human thioredoxin homodimers

T2 - Regulation by pH, role of aspartate 60, and crystal structure of the aspartate 60*[ asparagine mutant

AU - Andersen, John F.

AU - Sanders, David A R

AU - Gasdaska, John R.

AU - Weichsel, Andrzej

AU - Powis, Garth

AU - Montfort, William "Bill"

PY - 1997/11/18

Y1 - 1997/11/18

N2 - Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Gys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (K(app)) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 μM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pK(a) of 6.5. A similar value was obtained using gel filtration at pH 3.8 (K(app) = 164 μM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by ~2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Ash 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for K(app) suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.

AB - Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Gys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (K(app)) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 μM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pK(a) of 6.5. A similar value was obtained using gel filtration at pH 3.8 (K(app) = 164 μM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by ~2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Ash 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for K(app) suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.

UR - http://www.scopus.com/inward/record.url?scp=0030671351&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030671351&partnerID=8YFLogxK

U2 - 10.1021/bi971004s

DO - 10.1021/bi971004s

M3 - Article

C2 - 9369469

AN - SCOPUS:0030671351

VL - 36

SP - 13979

EP - 13988

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 46

ER -