Cardiac-specific Nkx2.5 homeodomain: conformational stability and specific DNA binding of Nkx2.5(C56S).
The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain of Drosophila when bound to the same specific DNA. The thermal unfolding of Nkx2.5(C56S) at pH 6.0 or 7.4 is a reversible, two-state process with unit cooperativity, as measured by differential scanning calorimetry (DSC) and far-UV circular dichroism. Adding 100 mM NaCl to Nkx2.5(C56S) at pH 7.4 increases T(m) from 44 to 54 +/- 0.2 degrees C and DeltaH from 34 to 45 +/- 2 kcal/mol (giving a DeltaC(p) of approximately 1.2 kcal K(-)(1) mol(-)(1) for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelike structures at <37 degrees C. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100 mM NaCl yield binding constants of 2-6 x 10(8) M(-)(1) from 10 to 37 degrees C and a stoichiometry of 1:1 for homeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5 is enthalpically controlled, and the temperature dependence of DeltaH gives a DeltaC(p) of -0.18 +/- 0.01 kcal K(-)(1) mol(-)(1). This corresponds to 648 +/- 36 A(2) of buried apolar surface upon Nkx2.5(C56S) binding duplex B-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA. Unbound NK-2 is more flexible than Nkx2.5.
Fodor
Elfrieda
E
Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-8012, USA.
Mack
James W
JW
Maeng
Jin-Soo
JS
Ju
Jeong-Ho
JH
Lee
Hyun Sook
HS
Gruschus
James M
JM
Ferretti
James A
JA
Ginsburg
Ann
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D004247:DNA / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C094797:vnd protein, Drosophila; D004247:DNA