DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers

J Mol Biol. 2000 Jan 28;295(4):719-27. doi: 10.1006/jmbi.1999.3406.


High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger proteins is mediated by both specific protein-base interactions and non-specific contacts between charged side-chains and the phosphate backbone. In addition, in DNA complexes of multiple zinc fingers, protein-protein interactions between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high- affinity binding, a highly specific DNA-induced helix capping involving residues in the linker sequence between fingers. Capping at the C terminus of the alpha-helix in each zinc finger, incorporating a consensus TGEKP linker sequence that follows each finger, provides substantial binding energy to the DNA complexes of zinc fingers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structures of each of the TGEKP linkers in these complexes can be superimposed on the linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structures from the zinc-finger-DNA complexes have been compared to the NMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 in the absence of DNA. This comparison reveals that additional stabilization likely arises in the DNA complexes from hydrogen bonding between the backbone amide of E3 and the side-chain O(gamma) of T1 in the linker. We suggest that these DNA-induced C-capping interactions provide a means whereby the multiple-finger complex, which must necessarily be domain-flexible in the unbound state as it searches for the correct DNA sequence, can be "snap-locked" in place once the correct DNA sequence is encountered. These observations provide a rationale for the high conservation of the TGEKP linker sequences in Cys(2)-His(2) zinc finger proteins.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Conserved Sequence
  • Crystallography, X-Ray
  • Cysteine
  • DNA / chemistry
  • DNA / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Histidine
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular / methods
  • Protein Structure, Secondary
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Transcription Factor TFIIIA
  • Transcription Factors / chemistry
  • WT1 Proteins
  • Zinc Fingers*


  • DNA-Binding Proteins
  • Transcription Factor TFIIIA
  • Transcription Factors
  • WT1 Proteins
  • Histidine
  • DNA
  • Cysteine