Robert T. Nolte

Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma.

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-γ ligand-binding domain (LBD), at 2.2u2009Å resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-γ. We also describe the ternary complex containing the PPAR-γ LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3u2009Å resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a ‘charge clamp’ that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-γ homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARα

Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR, which in turn recruit histone deacetylases to the chromatin. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-α ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn α-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of the co-repressor motif is further reinforced by the antagonist, which blocks the AF-2 helix from adopting the active position. Biochemical analyses and structure-based mutagenesis indicate that this mode of co-repressor binding is highly conserved across nuclear receptors.

X-ray crystal structures of the estrogen-related receptor-gamma ligand binding domain in three functional states reveal the molecular basis of small molecule regulation.

X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-γ (ERRγ) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERRγ LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERRγ seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERRγ LBD with 4-hydroxytamoxifen (4-OHT), and the ERRγ LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERRγ plus 4-OHT complex. The observed agonist-bound state contains the ERRγ LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERRγ by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix.

Structure of Rev-erbalpha bound to N-CoR reveals a unique mechanism of nuclear receptor-co-repressor interaction.

Repression of gene transcription by the nuclear receptor Rev-erbα plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor–co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbα ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel β-sheet with Rev-erbα, as well as an α-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbβ bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbα could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor–co-repressor interactions.

Allosteric Inhibition of Human Immunodeficiency Virus Integrase: LATE BLOCK DURING VIRAL REPLICATION AND ABNORMAL MULTIMERIZATION INVOLVING SPECIFIC PROTEIN DOMAINS*

Background: New antiviral agents bind to a site on HIV integrase protein also bound by the cellular protein LEDGF/p75. Results: Compound GSK1264 binds to this site, but it has surprising properties; it inhibits late during HIV replication, not early during integration, and it promotes abnormal multimerization. Conclusion: GSK1264 provides new insight into HIV replication. Significance: These observations inform the design of improved antiviral agents. HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for inxa0silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.

Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase.

The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.

Structure of Rev-erbα bound to N-CoR reveals a unique mechanism of nuclear receptor–co-repressor interaction

Repression of gene transcription by the nuclear receptor Rev-erbα plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor–co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbα ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel β-sheet with Rev-erbα, as well as an α-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbβ bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbα could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor–co-repressor interactions.