A Novel DNA Binding Mechanism for maf Basic Region-Leucine
Zipper Factors Inferred from a MafA–DNA Complex Structure and
Binding Specificities
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Abstract
MafA
is a proto-oncoprotein and is critical for insulin gene expression
in pancreatic β-cells. Maf proteins belong to the AP1 superfamily
of basic region-leucine zipper (bZIP) transcription factors. Residues
in the basic helix and an ancillary N-terminal domain, the Extended
Homology Region (EHR), endow maf proteins with unique DNA binding
properties: binding a 13 bp consensus site consisting of a core AP1
site (TGACTCA) flanked by TGC sequences and binding DNA stably as
monomers. To further characterize maf DNA binding, we determined the
structure of a MafA–DNA complex. MafA forms base-specific hydrogen
bonds with the flanking G<sub>–5</sub>C<sub>–4</sub> and central C<sub>0</sub>/G<sub>0</sub> bases, but not with the
core-TGA bases. However, in vitro binding studies utilizing a pulse–chase
electrophoretic mobility shift assay protocol revealed that mutating
either the core-TGA or flanking-TGC bases dramatically increases the
binding off rate. Comparing the known maf structures, we propose that
DNA binding specificity results from positioning the basic helix through
unique phosphate contacts. The EHR does not contact DNA directly but
stabilizes DNA binding by contacting the basic helix. Collectively,
these results suggest a novel multistep DNA binding process involving
a conformational change from contacting the core-TGA to contacting
the flanking-TGC bases