A sulfoglycolipid beta-sulfoquinovosyldiacylglycerol (betaSQDG) binds to Met1-Arg95 region of murine DNA polymerase lambda (Mmpol lambda) and inhibits its nuclear transit.
Beta-sulfoquinovosyldiacylglycerol (betaSQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase lambda (pol lambda). Here we identified a betaSQDG binding region within murine pol lambda (Mmpol lambda) using T7 phage display technology. We compared the binding intensity of betaSQDG with recombinant phages (phages lambda1-6) that displayed different segments of Mmpol lambda. The binding assay clearly showed that phage lambda1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to betaSQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific betaSQDG binding to Met1-Arg95 with a K(D) value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pollambda was inhibited in the presence of betaSQDG possibly due to binding of betaSQDG to NLS. These experiments clearly show that the binding region of betaSQDG within Mmpol lambda could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.
Takakusagi
K
K
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Takakusagi
Y
Y
Ohta
K
K
Aoki
S
S
Sugawara
F
F
Sakaguchi
K
K
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism*; D051189:Murinae; D019151:Peptide Library; D020349:Surface Plasmon Resonance
D006017:Glycolipids; D019151:Peptide Library; C013694:sulfoquinovosyl diglyceride; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta