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The activation of PPARgamma by oxidised fatty acids

4-oxo DHA

The nuclear receptor PPARgamma has important roles in adipogenesis and immune response as well as roles in both lipid and carbohydrate metabolism. Although synthetic agonists for PPARgamma are widely used as insulin sensitisers, the identity of the natural ligand(s) for PPARgamma is still not clear. Suggested natural ligands include 15-deoxy-D12,14-prostaglandin J2 and oxidised fatty acids such as 9-HODE and 13-HODE. Crystal structures of PPARgamma have revealed the mode of recognition for synthetic compounds.

Here we report structures of PPARgamma bound to oxidised fatty acids that are likely to be natural ligands for this receptor. These structures reveal that the receptor can (i) simultaneously bind two fatty acids and (ii) couple covalently with conjugated oxo fatty acids. Thermal stability and gene expression analyses suggest that such covalent ligands are particularly effective activators of PPARgamma and thus may serve as potent and biologically relevant ligands.

  • Itoh T, Fairall L, Amin K, Inaba Y, Szanto A, Balint BL, Nagy L, Yamamoto K, Schwabe JWR. Structural basis for the activation of PPARgamma by oxidised fatty acids. Nat Struct Mol Biol 2008, 15: p. 924-931.

The histone deacetylase activation domain from SMRT

4-helical structure of SMRT DAD

SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) are transcriptional corepressors that play an essential role in the regulation of development and metabolism.
This role is achieved, in part, through the recruitment of a key histone deacetylase (HDAC3), which is itself indispensable for cell viability.

The assembly of HDAC3 with the deacetylase activation domain (DAD) of SMRT and NCoR is required for activation of the otherwise inert deacetylase. The DAD comprises an N-terminal DAD-specific motif and a C-terminal SANT (SWI3_ADA2_NCoR_TFIIIB)-like domain.

We report here the solution structure of the DAD from SMRT, which reveals a four-helical structure. The DAD differs from the SANT (and MYB) domains in that (i) it has an additional N-terminal helix and (ii) there is a notable hydrophobic groove on the surface of the domain.

Structure-guided mutagenesis, combined with interaction assays, showed that residues in the vicinity of the hydrophobic groove are required for interaction with (and hence activation of) HDAC3. Importantly, one surface-exposed lysine is required for activation of HDAC3, but not for interaction. This lysine may play a uniquely important role in the mechanism of activating HDAC3.

  • Codina A, Love JD, Li Y, Lazar MA, Neuhaus D, Schwabe JWR. Structural insights into the interaction and activation of HDAC3 by nuclear receptor co-repressors. Proc Natl Acad Sci USA 2005, 102: p. 6009-6014.

Novel co-regulator binding surface on Nurr1

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The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity.

This volume is instead filled with bulky hydrophobic side chains. Furthermore the 'canonical' non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD.

We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors.

  • Codina A, Benoit G, Gooch JT, Neuhaus D, Perlmann T, Schwabe JW. Identification of a novel co-regulator interaction surface on the ligand binding domain of Nurr1 using NMR footprinting. J Biol Chem 2004, 279: p. 53338-45.

The SPOC Domain from SHARP

Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterised by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain.

The specific biological role of the SPOC domain (Spen paralog and ortholog C-terminal domain), and hence the common function of Spen proteins, has been unclear to date. The Spen protein SHARP (SMRT/HDAC1-associated repressor protein) was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-Jkappa signaling pathways. We have determined the 1.8 A crystal structure of the SPOC domain from SHARP. This structure shows that essentially all of the conserved surface residues map to a positively charged patch.

Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencing mediator for retinoid and thyroid receptors/nuclear receptor corepressor). We demonstrate that this interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors and that Spen proteins play an essential role in the repression complex.

  • Ariyoshi M, Schwabe JW. A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling. Genes Dev, 2003. 17: p. 1909-20.

Nuclear receptor dynamics

Graph of fluorescence anistropy against time

Nuclear receptors are transcription factors that activate gene expression in response to ligands. The C-terminal helix (helix 12) of the ligand-binding domain plays a critical role in the activation mechanism. When bound to activating ligands, helix 12 adopts a conformation that promotes the binding of co-activator proteins. Helix 12 also adopts this 'active' position in several ligand-free structures, raising questions as to the exact role of helix 12.

We proposed that the dynamic properties of helix 12 may be critical for the activation mechanism and, to test this, have used fluorescence anisotropy techniques to directly monitor the mobility of helix 12 in PPARgamma. Our results suggest that helix 12 is significantly more mobile than the main body of the protein. Upon ligand binding, helix 12 shows reduced mobility, accounting for its role as a molecular switch.

We also show that natural mutations in human PPARgamma, associated with severe insulin resistance and diabetes mellitus, exhibit perturbations in the dynamic behavior of helix 12. Our findings provide the first direct observations of the mobility of helix 12 and suggest that the dynamic properties of this helix are key to the regulation of transcriptional activity.

  • Kallenberger BC, Love JD, Chatterjee VK, Schwabe JW. A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease. Nat Struct Biol, 2003. 10: p. 136-40.

Specific ligands for RXR

structure of hRXRbeta

Ligands that specifically target retinoid-X receptors (RXRs) are emerging as potentially powerful therapies for cancer, diabetes,and the lowering of circulatory cholesterol. To date, RXR has only been crystallised in the absence of ligand or with the promiscuous ligand 9-cis retinoic acid, which also activates retinoic acid receptors.

Here we present the structure of hRXRbeta in complex with the RXR-specific agonist LG100268 (LG268). The structure clearly reveals why LG268 is specific for the RXR ligand binding pocket and will not activate retinoic acid receptors.

Intriguingly, in the crystals, the C-terminal 'activation' helix (AF-2/helix H12) is trapped in a novel position not seen in other nuclear receptor structures such that it does not cap the ligand binding cavity. Mammalian two-hybrid assays indicate that LG268 is unable to release co-repressors from RXR unless co-activators are also present. Together these findings suggest that RXR ligands may be inefficient at repositioning helix H12.

  • Love JD, Gooch JT, Benko S, Li C, Nagy L, Chatterjee VK, Evans RM, Schwabe JW. The structural basis for the specificity of retinoid-X receptor-selective agonists: new insights into the role of helix H12. J Biol Chem, 2002. 277: p. 11385-91.

An unexpected ligand for Ultraspiracle

structure of USP ligand-binding domain

Ultraspiracle (USP) is the invertebrate homologue of the mammalian retinoid X receptor (RXR). RXR plays a uniquely important role in differentiation, development, and homeostasis through its ability to serve as a heterodimeric partner to many other nuclear receptors.

RXR is able to influence the activity of its partner receptors through the action of the ligand 9-cis retinoic acid. In contrast to RXR, USP has no known high-affinity ligand and is thought to be a silent component in the heterodimeric complex with partner receptors such as the ecdysone receptor.

Here we report the 2.4-Å crystal structure of the USP ligand-binding domain. The structure shows that a conserved sequence motif found in dipteran and lepidopteran USPs, but not in mammalian RXRs, serves to lock USP in an inactive conformation. It also shows that USP has a large hydrophobic cavity, implying that there is almost certainly a natural ligand for USP. This cavity is larger than that seen previously for most other nuclear receptors. Intriguingly, this cavity has partial occupancy by a bound lipid, which is likely to resemble the natural ligand for USP.

  • Clayton GM, Peak-Chew SY, Evans RM, Schwabe JW. The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation. Proc Natl Acad Sci USA, 2001. 98: p. 1549-54.

How co-repressors bind nuclear receptors


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The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity. A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator. Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs.

We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release. Interestingly, the motif contains a hydrophobic core (PhixxPhiPhi) similar to that found in NR coactivators. Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association. These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site.

  • Nagy L, Kao HY, Love JD, Li C, Banayo E, Gooch JT, Chatterjee VKK, Evans RM, Schwabe JW. Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev, 1999. 13: p. 3209-16.
  • Benko S, Love JD, Beládi M, Schwabe, JWR, Nagy L. Molecular determinants of the balance between co-repressor and co-activator recruitment to the retinoic acid receptor: identification of residues that bias helix 12 positioning. J. Biol. Chem., 2003. 278: p. 43797-806.

DNA-binding by the oestrogen receptor

The nuclear hormone receptors are a superfamily of ligand-activated DNA-binding transcription factors. We have determined the crystal structure (at 2.4Å) of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA.

The protein binds as a symmetrical dimer to its palindromic binding site consisting of two 6 bp consensus half sites with three intervening base pairs. This structure reveals how the protein recognises its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs. Since all nuclear hormone receptors recognise one or the other of these two consensus half site sequences, this recognition mechanism applies generally to the whole receptor family.

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  • Schwabe JW, Chapman L, Finch JT, Rhodes D. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell, 1993. 75: p. 567-78.

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