68 BIOCHEMISTRY FUNDAMENTALS
pair subsite. Each of the zinc fi ngers uses arginine, histidine, and aspartic acid
amino acid residues from the N - terminal portion of an α - helix to make contact
the bases in the major DNA groove. Most of the zinc - fi nger – DNA contacts
are made with the guanine - rich strand of the DNA. The N ε 2 nitrogen atoms of
his ligands coordinate to the Zn(II) ion; for example, the his53 NZnε 22 −^2 + bond
distance is 2.00 Å. From the same histidine ligands, the N δ 1 nitrogen atom
hydrogen bonds to phosphodiester oxygens of DNA; for example, the his53
Nδ 1 – O 2P H - bond distance is 2.66 Å. In Figure 2.22 , two N δ 1 – O 2P hydrogen bonds
are indicated by dot surfaces.
Earlier experiments had already shown that zinc - fi nger protein design could
test DNA binding specifi city. For instance, Desjarlais and Berg in 1993 had
designed three zinc - fi nger proteins with different DNA binding specifi cities.^36
The design strategy combined a consensus zinc - fi nger framework sequence
with previously characterized recognition regions such that the specifi city of
each protein was predictable. The fi rst protein consisted of three identical zinc
fi ngers, each of which was expected to recognize the subsite GCG. The designed
protein binds specifi cally to the sequence 5 ′ - GCG - GCG - GCG - 3 ′ with a dis-
sociation constant of approximately 11 μ M. The second protein had three zinc
fi ngers with different predicted preferred subsites and would bind to the pre-
dicted recognition site 5 ′ - GGG - GCG - GCT - 3 ′ with a dissociation constant of
2 nM. A permuted version of the second protein was also constructed and
shown to preferentially recognize the corresponding permuted site 5 ′ - GGG -
Figure 2.22 Zinc - fi nger – DNA interactions for PDB: 1ZAA as described in reference
31 and the text. Visualized using The PyMOL Molecular Graphics System and Chem-
Draw Ultra, version 10.0. (Printed with permission of Delano Scientifi c, LLC and
CambridgeSoft Corporation.) (See color plate)
cys40
cys37his53 his57G4his25G7zinc1zinc2zinc3PDB: 1ZAA.
DNA Strand in orange ribbon.
Protein in green cartoon.