The Essential Role of Prolines and Their Conformation in Allosteric Regulation of Kaiso Zinc Finger DNA-Binding Activity by the Adjacent C-Terminal Loop

Abstract

Kaiso is a methyl-DNA-binding protein containing three C2H2 zinc fingers with a C-terminal extension that participates in DNA binding. The linker between the last zinc finger and the DNA-binding portion of the extension contains two prolines that are highly conserved in vertebrates and in cognate ZBTB4 and ZBTB38 proteins. Prolines provide chain rigidity and can exist in cis and trans conformations that can be switched by proline isomerases, affecting protein function. We found that substitution of the conserved proline P588, but not of P577, to alanine, negatively affected KaisoDNA-binding according to molecular dynamics simulation and in vitro DNA-binding assays. Molecular dynamics simulations of the Kaiso DNA-binding domain with P588 either substituted to alanine or switched to the cis-conformation revealed similar alterations in the H-bonding network and uncovered allosteric effects leading to structural rearrangements in the entire domain that resulted in the weakening of DNA-binding affinity. The substitution of proline with a large hydrophobic residue led to the same negative effects despite its ability to partially rescue the intrinsic DNA-binding activity of the C-terminal loop. Thus, the presence of the C-terminal extension and cis-conformation of proline residues are essential for efficient Kaiso–DNA binding, which likely involves intramolecular tension squeezing the DNA chain.

Keywords

• DNA methylation
• C2H2 zinc finger proteins
• molecular dynamics
• methyl(DNA)-binding proteins