PROPOSED MECHANISM FOR THE STABILIZATION OF NUCLEAR RECEPTOR DNA-BINDING VIA PROTEIN DIMERIZATION

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.