Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM

Masanori Osawa, Alexandra Dace, Kit I. Tong, Aswani Valiveti, Mitsuhiko Ikura, James B. Ames

Research output: Contribution to journalArticle

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Abstract

DREAM (calsenilin/KChlP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca2+ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca2+ binding to the various mutants revealed that, in the absence of Mg2+, Ca2+ binds independently and se-quentially to EF-3 (ΔH = -2.4 kcal/mol), EF-4 (ΔH = +5.2 kcal/mol), and EF-2 (ΔH = +1 kcal/mol). By contrast, only two Ca2+ bind to DREAM in the presence of physiologi-cal levels of Mg2+ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg2+. ITC measurements demonstrate that one Mg2+ binds enthalpically with high affinity (Kd = 13 μM and ΔH = -0.79 kcal/mol) and two or more Mg2+ bind entropically in the millimolar range. Size-exclusion chromatograpliy studies revealed that Mg2+ stabilizes DREAM as a monomer, whereas Ca2+ induces protein dinierization. Electrophoretic mobility shift assays indicated that Mg2+ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca2+ levels, suggesting that binding of a single Ca 2+ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca2+ and Mg2+ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg2+ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca 2+-induced protein dimerization disrupts DNA binding.

Original languageEnglish
Pages (from-to)18008-18014
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number18
DOIs
Publication statusPublished - 2005 May 6
Externally publishedYes

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Dimerization
Peptide Elongation Factor 2
EF Hand Motifs
DNA
Response Elements
fos Genes
Calorimetry
Titration
Kv Channel-Interacting Proteins
Genes
Protein Multimerization
Electrophoretic mobility
Calcium-Binding Proteins
Electrophoretic Mobility Shift Assay
Transcription
Structural analysis
Molten materials
Assays
Proteins
Monomers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM. / Osawa, Masanori; Dace, Alexandra; Tong, Kit I.; Valiveti, Aswani; Ikura, Mitsuhiko; Ames, James B.

In: Journal of Biological Chemistry, Vol. 280, No. 18, 06.05.2005, p. 18008-18014.

Research output: Contribution to journalArticle

Osawa, Masanori ; Dace, Alexandra ; Tong, Kit I. ; Valiveti, Aswani ; Ikura, Mitsuhiko ; Ames, James B. / Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 18. pp. 18008-18014.
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abstract = "DREAM (calsenilin/KChlP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca2+ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca2+ binding to the various mutants revealed that, in the absence of Mg2+, Ca2+ binds independently and se-quentially to EF-3 (ΔH = -2.4 kcal/mol), EF-4 (ΔH = +5.2 kcal/mol), and EF-2 (ΔH = +1 kcal/mol). By contrast, only two Ca2+ bind to DREAM in the presence of physiologi-cal levels of Mg2+ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg2+. ITC measurements demonstrate that one Mg2+ binds enthalpically with high affinity (Kd = 13 μM and ΔH = -0.79 kcal/mol) and two or more Mg2+ bind entropically in the millimolar range. Size-exclusion chromatograpliy studies revealed that Mg2+ stabilizes DREAM as a monomer, whereas Ca2+ induces protein dinierization. Electrophoretic mobility shift assays indicated that Mg2+ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca2+ levels, suggesting that binding of a single Ca 2+ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca2+ and Mg2+ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg2+ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca 2+-induced protein dimerization disrupts DNA binding.",
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AU - Dace, Alexandra

AU - Tong, Kit I.

AU - Valiveti, Aswani

AU - Ikura, Mitsuhiko

AU - Ames, James B.

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N2 - DREAM (calsenilin/KChlP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca2+ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca2+ binding to the various mutants revealed that, in the absence of Mg2+, Ca2+ binds independently and se-quentially to EF-3 (ΔH = -2.4 kcal/mol), EF-4 (ΔH = +5.2 kcal/mol), and EF-2 (ΔH = +1 kcal/mol). By contrast, only two Ca2+ bind to DREAM in the presence of physiologi-cal levels of Mg2+ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg2+. ITC measurements demonstrate that one Mg2+ binds enthalpically with high affinity (Kd = 13 μM and ΔH = -0.79 kcal/mol) and two or more Mg2+ bind entropically in the millimolar range. Size-exclusion chromatograpliy studies revealed that Mg2+ stabilizes DREAM as a monomer, whereas Ca2+ induces protein dinierization. Electrophoretic mobility shift assays indicated that Mg2+ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca2+ levels, suggesting that binding of a single Ca 2+ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca2+ and Mg2+ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg2+ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca 2+-induced protein dimerization disrupts DNA binding.

AB - DREAM (calsenilin/KChlP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca2+ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca2+ binding to the various mutants revealed that, in the absence of Mg2+, Ca2+ binds independently and se-quentially to EF-3 (ΔH = -2.4 kcal/mol), EF-4 (ΔH = +5.2 kcal/mol), and EF-2 (ΔH = +1 kcal/mol). By contrast, only two Ca2+ bind to DREAM in the presence of physiologi-cal levels of Mg2+ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg2+. ITC measurements demonstrate that one Mg2+ binds enthalpically with high affinity (Kd = 13 μM and ΔH = -0.79 kcal/mol) and two or more Mg2+ bind entropically in the millimolar range. Size-exclusion chromatograpliy studies revealed that Mg2+ stabilizes DREAM as a monomer, whereas Ca2+ induces protein dinierization. Electrophoretic mobility shift assays indicated that Mg2+ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca2+ levels, suggesting that binding of a single Ca 2+ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca2+ and Mg2+ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg2+ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca 2+-induced protein dimerization disrupts DNA binding.

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