Structure of a 13-fold superhelix (almost) determined from first principles.

Nuclear hormone receptors are cytoplasm-based transcription factors that bind a ligand, translate to the nucleus and initiate gene transcription in complex with a co-activator such as TIF2 (transcriptional intermediary factor 2). For structural studies the co-activator is usually mimicked by a peptide of circa 13 residues, which for the largest part forms an α-helix when bound to the receptor. The aim was to co-crystallize the glucocorticoid receptor in complex with a ligand and the TIF2 co-activator peptide. The 1.82 Å resolution diffraction data obtained from the crystal could not be phased by molecular replacement using the known receptor structures. HPLC analysis of the crystals revealed the absence of the receptor and indicated that only the co-activator peptide was present. The self-rotation function displayed 13-fold rotational symmetry, which initiated an exhaustive but unsuccessful molecular-replacement approach using motifs of 13-fold symmetry such as α- and β-barrels in various geometries. The structure was ultimately determined by using a single α-helix and the software ARCIMBOLDO, which assembles fragments placed by PHASER before using them as seeds for density modification model building in SHELXE. Systematic variation of the helix length revealed upper and lower size limits for successful structure determination. A beautiful but unanticipated structure was obtained that forms superhelices with left-handed twist throughout the crystal, stabilized by ligand interactions. Together with the increasing diversity of structural elements in the Protein Data Bank the results from TIF2 confirm the potential of fragment-based molecular replacement to significantly accelerate the phasing step for native diffraction data at around 2 Å resolution

Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ∼5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Although the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2, 3, 4, 5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5, 6, and is therefore not well understood. Here, we show that stiffness topography7 with sharp atomic force microscopy tips can generate nanoscale cross-sections of the NPC. The cross-sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy2, 3, 4, 5. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel

Engineering of a Water-Soluble Plant Cytochrome P450, CYP73A1, and NMR-Based Orientation of Natural and Alternate Substrates in the Active Site

CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L(–1) yeast culture. This water-soluble enzyme was suitable for (1)H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and (1)H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the (1)H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle

Enhancing the stability and solubility of the glucocorticoid receptor ligand-binding domain by high-throughput library screening

The human glucocorticoid receptor ligand-binding domain (hGR-LBD) is an important drug target for the treatment of various diseases. However, the low intrinsic stability and solubility of hGR-LBD have rendered its purification and biophysical characterization difficult. In order to overcome these problems, we have stabilized hGR-LBD by a combination of random mutagenesis and high-throughput screening using fluorescence-activated cell sorting (FACS) with enhanced green fluorescent protein (eGFP) as folding reporter. Two plasmid-encoded gene libraries of hGR-LBD fused to the egfp gene were expressed in Escherichia coli, followed by eight rounds of FACS screening, in each of which 10(8) cells were analyzed. The hgr-lbd mutants isolated by this approach contained numerous amino acid exchanges, and four beneficial ones (A605V, V702A, E705G, and M752T) were followed up in detail. Their characterization showed that the fluorescence of hGR-LBD-eGFP fusions is correlated linearly with the stability and solubility of hGR-LBD in the absence of eGFP. When combined, the four exchanges increased the thermal stability of hGR-LBD by more than 8 °C and enhanced its purification yield after expression in E. coli by about 26-fold. The introduction of three beneficial exchanges into the homologous ligand-binding domain of mouse enabled its X-ray structure determination at high resolution, which showed how the exchanges stabilize the protein and revealed atomic details that will guide future drug design. Our results demonstrate that large eGFP fusion libraries can be screened by FACS with extreme sensitivity and efficiency, yielding stabilized eukaryotic proteins suitable for biophysical characterization and structure determination

Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat

Electronic Supplementary : Material Supplementary material is available to authorised users in the online version of this article at http://dx.doi.org/10.1007/s11103-006-9028-8.International audienceA burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs—receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Gα when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Gα, RGS domain binding consequently accelerates Gα-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Gα substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Gα selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Gα complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Gα substrate, suggests that unique structural determinants specific to particular RGS protein/Gα pairings exist and could be used to achieve selective inhibition by small molecules