Sequence-specific recognition of DNA by hydrophobic, alanine-rich mutants of the basic region/leucine zipper motif investigated by fluorescence anisotropy.
We generated minimalist proteins capable of sequence-specific, high-affinity binding of DNA to probe how proteins are used and can be used to recognize DNA. In order to quantify binding affinities and specificities in our protein-DNA system, we used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) domain to DNA duplexes containing target sites AP-1 (5'-TGACTCA-3') or ATF/CREB (5'-TGACGTCA-3'). We simplified the alpha-helical bZIP molecular recognition scaffold by alanine substitution: 4A, 11A, and 18A contain four, eleven, and eighteen alanine mutations in their DNA-binding basic regions, respectively. DNase I footprinting analysis demonstrates that all bZIP mutants retain the sequence-specific DNA-binding function of native GCN4 bZIP. Titration of fluorescein-labeled oligonucleotide duplexes with increasing amounts of protein yielded low nanomolar dissociation constants for all bZIP mutants in complex with the AP-1 and ATF/CREB sites: binding to the nonspecific control duplex was > 1000-fold weaker. Remarkably, the most heavily mutated protein 18A, containing 24 alanines in its 27-residue basic region, still binds AP-1 and ATF/CREB with dissociation constants of 15 and 7.8 nM, respectively. Similarly, wild-type bZIP binds these sites with K(d) values of 9.1 and 14 nM. 11A also displays low nanomolar dissociation constants for AP-1 and ATF/CREB, while 4A binds these sites with approximately 10-fold weaker K(d) values. Thus, both DNA-binding specificity and affinity are maintained in all our bZIP derivatives. This Ala-rich scaffold may be useful in design and synthesis of small alpha-helical proteins with desired DNA-recognition properties capable of serving as therapeutics targeting transcription.
Bird
Gregory H
GH
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Lajmi
Ajay R
AR
Shin
Jumi A
JA
D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D001345:Autoradiography; D004247:DNA / Q000378:metabolism*; D016172:DNA Fingerprinting; D003850:Deoxyribonuclease I; D005454:Fluorescence Polarization; D016350:Leucine Zippers / Q000502:physiology*; D009154:Mutation
D004247:DNA; D003850:Deoxyribonuclease I; D000409:Alanine