Zinc finger protein-DNA recognition

Zinc finger proteins are transcription factors containing three zinc-finger motifs, each of which has the ability to recognize and bind to a nucleotide triplet. It is known that three residue positions on each motif (-1, +3 and +6 relative to the α-helix in the motif, defined as our recognition helix) are decisive in determining specificity of DNA recognition (1). Hydrogen bonds between the amino acids and nucleotides at the protein-DNA interface are known to be a major contributor to specificity (2).

 

Principle of prediction

The interfacial hydrogen bond energy (IHBE), defined as the energy of hydrogen bonding at the protein-DNA interface of the amino acid side chains in the recognition helix and nucleotide side-chains in the binding triplet is the relative score used to rank recognition helix preferences for each nucleotide triplet at a particular finger position. These scores were calculated in advance for all possible mutant Protein Data Bank (PDB) structures of Zif268 (1AAY) using our predictive algorithm describd in the manuscript and can be retrieved using the interface here.

To compute IHBE for approach1 (Modular mode) and approach2 (Synergistic mode):

  1. A temporary PDB mutant structure was generated (eventually for all finger × triplet × recognition helix combinations) using our algorith for the two approaches respectively.The mutations in approach 1 & 2 were using a pool of most commonly occuring amino acids extracted from literature.The consensus pool of amino acids have been tabulated:

    2.  

  2. Potential hydrogen bonding pairs involving the recognition helix amino acid binding site nucleotide side-chains were determined, assuming donor and acceptor atoms are within 350 pm of each other (4).
  3. IHBE was calculated using equation given in the manuscript.

Recognition helix preferences are arranged in decreasing order of IHBE for each finger-triplet combination.
Approach 3 is also a predictive algorithm designed for modular mode of binding accepting all (20)amino acid mutations unlike the consensus pool used in approach 1 & 2, but we have chosen to refrain from uploading the algorithm for predictive purpose as the results leave scope for improvement.

The two modes of bindng incorporated by our predictive algorithm

 

 

Using the recognition helix output

A recognition helix set (for the three fingers) is inserted in the following positions in the backbone sequence of Zif268.

 

Finger

Position in protein chain (1AAY_A)


-1

2

3

6

1

118

120

121

124

2

146

148

149

152

3

174

176

177

180


References

  1. Klug, A. (2010) The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev Biochem 79, 213–31.

  2. Wolfe, S. A., Nekludova, L., and Pabo, C. O. (2000) DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29, 183–212.

  3. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Improved methods for build- ing protein models in electron density maps and the location of errors in these models. Acta Crystallographica Section A 47, 110–119.

  4. Hydrogen Bonding, Glossary, www.proteinexplorer.org.

  5. Wendler, K., Thar, J., Zahn, S., and Kirchner, B. (2010) Estimating the Hydrogen Bond Energy . The Journal of Physical Chemistry A 114, 9529–9536.