Exploring the recognition of quadruplex DNA by an engineered Cys2-His2 zinc finger protein.
We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.
Ladame
Sylvain
S
University Chemical Laboratories, University of Cambridge, UK.
Schouten
James A
JA
Roldan
Jose
J
Redman
James E
JE
Neidle
Stephen
S
Balasubramanian
Shankar
S
D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D054856:G-Quadruplexes; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D011994:Recombinant Proteins; D006639:Histidine; D004247:DNA; D003545:Cysteine