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 homodimers. We recently showed that the putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. In this report, the role of the LBD in DNA binding by HNF-4 is further investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 showed that dimerization via the DBD was sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD was not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increased the half-life of the complex by at least 100-fold. Circular permutation analysis showed that full-length HNF-4 bent DNA by approximately 80 degrees while the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other constructs indicated that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. Coimmunoprecipitation studies, on the other hand, showed that dimerization 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 an energetically unfavorable single-step event. Implications of this one-step model for other nuclear receptors are discussed.
Jiang
G
G
Environmental Toxicology Graduate Program, University of California, Riverside 92521, USA.
Lee
U
U
Sladek
F M
FM
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006207:Half-Life; D051557:Hepatocyte Nuclear Factor 4; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA