Recognition properties of a sequence-specific DNA binding antibody.
A sequence-specific DNA-binding antibody was previously generated by incorporating a 17 amino acid alpha-helix from the DNA-binding domain of the transcription factor TFEB into the HCDR3 site of a recombinant human Fab fragment. The recombinant DNA-binding antibody, called Fab-E box, binds the TFEB recognition sequence CACGTG (an E box site) with a 5-10-fold lower affinity than TFEB. Here, we have determined the precise kinetics of interaction of Fab-E box with DNA and show that the lower affinity of Fab-E box relative to TFEB for E box DNA is due to a higher dissociation rate. DNase I protection assays show Fab-E box physically interacts with one half-site of the E box. Additional DNA target sites of Fab-E box were identified by DNase I protection assays. A compilation of these binding sites indicates that the recognition elements for Fab-E box binding include a half-site of the E box, CAW, with an 8 bp consensus sequence identified as YNYYCAWW. Thus, the DNA determinants for Fab-E box recognition extend beyond one-half site of the E box sequence, with preferences for pyrimidines and A+T-rich sequences in the 5' and 3' outer regions of the binding site, respectively. Apparent dissociation constants of Fab-E box for a subset of these target DNA sequences are 5-10-fold greater than the DNA-binding affinity of the antibody with the E box site. Therefore, these results identify important DNA specificity determinants for high-affinity binding by Fab-E box.
LeBlanc
J F
JF
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
McLane
K E
KE
Parren
P W
PW
Burton
D R
DR
Ghazal
P
P
D000918:Antibody Specificity / Q000235:genetics; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D004247:DNA / Q000276:immunology* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs / Q000235:genetics / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D014157:Transcription Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D009363:Neoplasm Proteins; C064972:TFEB protein, human; D014157:Transcription Factors; D004247:DNA