The retargeting of protein–DNA specificity, outside
of extremely modular DNA binding proteins such
as TAL effectors, has generally proved to be quite
challenging. Here, we describe structural analyses
of five different extensively retargeted variants of a
single homing endonuclease, that have been shown
to function efficiently in ex vivo and in vivo applications.
The redesigned proteins harbor mutations
at up to 53 residues (18%) of their amino acid sequence,
primarily distributed across the DNA binding
surface, making them among the most signifi-
cantly reengineered ligand-binding proteins to date.
Specificity is derived from the combined contributions
of DNA-contacting residues and of neighboring
residues that influence local structural organization.
Changes in specificity are facilitated by the
ability of all those residues to readily exchange both
form and function. The fidelity of recognition is not
precisely correlated with the fraction or total number
of residues in the protein–DNA interface that are
actually involved in DNA contacts, including directional
hydrogen bonds. The plasticity of the DNArecognition
surface of this protein, which allows substantial
retargeting of recognition specificity without
requiring significant alteration of the surrounding
protein architecture, reflects the ability of the corresponding
genetic elements to maintain mobility and
persistence in the face of genetic drift within potential
host target sites.