PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of anti-diabetes drugs including rosiglitazone and pioglitazone1. These drugs are full classical agonists for this nuclear receptor, but recent data has shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk52. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent anti-diabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of anti-diabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.

Binding to helix 12 of the ligand-binding domain of PPARgamma is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPARgamma with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPARgamma transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.

Human Proliferating Cellular Nuclear Antigen (hPCNA), a member of the sliding clamp family of proteins, makes specific protein-protein interactions with DNA replication and repair proteins through a small peptide motif termed the PCNA-interacting protein, or PIP-box. We solved the structure of hPCNA bound to PIP-box-containing peptides from the p66 subunit of the human replicative DNA polymerase-delta (452-466) at 2.6 A and of the flap endonuclease (FEN1) (331-350) at 1.85 A resolution. Both structures demonstrate that the pol-delta p66 and FEN1 peptides interact with hPCNA at the same site shown to bind the cdk-inhibitor p21(CIP1). Binding studies indicate that peptides from the p66 subunit of the pol-delta holoenzyme and FEN1 bind hPCNA from 189- to 725-fold less tightly than those of p21. Thus, the PIP-box and flanking regions provide a small docking peptide whose affinities can be readily adjusted in accord with biological necessity to mediate the binding of DNA replication and repair proteins to hPCNA.

Understanding how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by significant misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems common to steroid hormone receptors are circumvented by a mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the first structure of an apo steroid receptor, revealing a ligand-accessible channel, allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFκB selective signaling through ER, revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define the structural features of signaling specificity that would not be apparent in a single structure.

SummaryThe vitamin D receptor (VDR) functions as an obligate heterodimer with the retinoid X receptor (RXR). These nuclear receptors (NRs) are multidomain proteins and it is unclear how various domains interact with one another within the NR heterodimer. Here we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA binding domains (DBDs), including the coactivator binding surfaces of both coreceptors, and the sequence of the DNA response element can specify the dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that ligand itself may play a role in DNA recognition. These data suggest a mechanism by which NRs can display promoter-specific activity and impart differential effects on various target genes, which provides mechanistic insight for the function of selective NR modulators.