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

High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger p roteins is mediated by both specific protein-base interactions and nonspeci fic contacts between charged side-chains and the phosphate backbone. Ln add ition, in DNA complexes of multiple zinc fingers, protein-protein interacti ons between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high-affinity binding, a highly s pecific 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 fi ngers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor s uppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structure s of each of the TGEKP linkers in these complexes can be superimposed on th e linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structu res from the zinc-finger-DNA complexes have been compared to the NMR struct ure 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 E 3 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 on ce the correct DNA sequence is encountered. These observations provide a ra tionale for the high conservation of the TGEKP linker sequences in Cys(2)-H is(2) zinc finger proteins. (C) 2000 Academic Press.