Interaction between CK2α and CK2β, the Subunits of Protein Kinase CK2

Thermodynamic Contributions of Key Residues on the CK2α Surface

J Raaf, N Bischoff, K Kloppfleisch, E Brunstein, Birgitte Brinkmann Olsen, G Vilk, D Litchfield, Olaf-Georg Issinger, K Niefind

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.
Original languageEnglish
JournalBiochemistry
Volume50
Issue number4
Pages (from-to)512-22
Number of pages11
ISSN0006-2960
DOIs
Publication statusPublished - 1. Feb 2011

Fingerprint

Casein Kinase II
Holoenzymes
Calorimetry
Titration
Phosphotransferases
Thermodynamics
Amino Acids
Mutagenesis
Mutation
Alanine
Dimers
Ionization
Names
Enthalpy
Catalytic Domain
Buffers
Substitution reactions
Scanning
Molecules
Pharmaceutical Preparations

Keywords

  • Alanine
  • Amino Acid Substitution
  • Casein Kinase II
  • Humans
  • Hydrogen-Ion Concentration
  • Isoenzymes
  • Leucine
  • Membrane Proteins
  • Peptide Fragments
  • Phenylalanine
  • Temperature
  • Thermodynamics

Cite this

Raaf, J ; Bischoff, N ; Kloppfleisch, K ; Brunstein, E ; Olsen, Birgitte Brinkmann ; Vilk, G ; Litchfield, D ; Issinger, Olaf-Georg ; Niefind, K. / Interaction between CK2α and CK2β, the Subunits of Protein Kinase CK2 : Thermodynamic Contributions of Key Residues on the CK2α Surface. In: Biochemistry. 2011 ; Vol. 50, No. 4. pp. 512-22.
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abstract = "The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.",
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author = "J Raaf and N Bischoff and K Kloppfleisch and E Brunstein and Olsen, {Birgitte Brinkmann} and G Vilk and D Litchfield and Olaf-Georg Issinger and K Niefind",
year = "2011",
month = "2",
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Interaction between CK2α and CK2β, the Subunits of Protein Kinase CK2 : Thermodynamic Contributions of Key Residues on the CK2α Surface. / Raaf, J; Bischoff, N; Kloppfleisch, K; Brunstein, E; Olsen, Birgitte Brinkmann; Vilk, G; Litchfield, D; Issinger, Olaf-Georg; Niefind, K.

In: Biochemistry, Vol. 50, No. 4, 01.02.2011, p. 512-22.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Interaction between CK2α and CK2β, the Subunits of Protein Kinase CK2

T2 - Thermodynamic Contributions of Key Residues on the CK2α Surface

AU - Raaf, J

AU - Bischoff, N

AU - Kloppfleisch, K

AU - Brunstein, E

AU - Olsen, Birgitte Brinkmann

AU - Vilk, G

AU - Litchfield, D

AU - Issinger, Olaf-Georg

AU - Niefind, K

PY - 2011/2/1

Y1 - 2011/2/1

N2 - The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.

AB - The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.

KW - Alanine

KW - Amino Acid Substitution

KW - Casein Kinase II

KW - Humans

KW - Hydrogen-Ion Concentration

KW - Isoenzymes

KW - Leucine

KW - Membrane Proteins

KW - Peptide Fragments

KW - Phenylalanine

KW - Temperature

KW - Thermodynamics

U2 - 10.1021/bi1013563

DO - 10.1021/bi1013563

M3 - Journal article

VL - 50

SP - 512

EP - 522

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 4

ER -