Gq. Jiang et al., PROPOSED MECHANISM FOR THE STABILIZATION OF NUCLEAR RECEPTOR DNA-BINDING VIA PROTEIN DIMERIZATION, Molecular and cellular biology, 17(11), 1997, pp. 6546-6554
Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear
receptors that exist in solution and bind DNA exclusively as homodimer
s. We recently showed that the putative ligand binding domain (LED) of
HNF-4 is responsible far dimerization in solution and prevents hetero
dimerization with other receptors. In this report, the role of the LED
in DNA binding by HNF-4 is further investigated by using electrophore
tic mobility shift analysis. A comparison of constructs containing eit
her the DNA binding domain (DBD) alone or the DBD plus the LED of HNF-
4 showed that dimerization via the DBD was sufficient to provide nearl
y the full DNA binding affinity of the full-length HNF-4. In contrast,
dimerization via the DBD was not sufficient to produce a stable prote
in-DNA complex, whereas dimerization via the LED increased the hair-li
fe of the complex by al feast 100-fold, Circular permutation analysis
showed that full-length HNF-4 bent DNA by approximately 80 degrees whi
le the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other
constructs indicated that the increase in stability afforded by the L
BD could be explained only partially by an increased ability to bend D
NA. Coimmunoprecipitation studies, on the other hand, showed that dime
rization via the LBD produced a protein-protein complex that was much
more stable than the corresponding protein-DNA complex, These results
led us to propose a model in which dimerization via the LBD stabilizes
the receptor on DNA by converting an energetically favorable two-step
dissociation event into are energetically unfavorable single-step eve
nt, Implications of this one-step model for other nuclear receptors ar
e discussed.