Multiple Binding Modes between HNF4α and the LXXLL Motifs of PGC-1α Lead to Full Activation

Hepatocyte nuclear factor 4 alpha (HNF4 alpha) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4 alpha interacts, peroxisome proliferation-activated receptor gamma(PPAR gamma) coactivator 1 alpha(PGC-1 alpha) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1 alpha recruitment, we have determined the crystal structure of HNF4 alpha in complex with a fragment of PGC-1 alpha containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4 alpha toward the LXXLL motifs of PGC-1 alpha could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.