Structural studies of herbicide detoxifying enzymes

Glutathione S-transferases (GSTs) [EC 2.5.1.18] are a ubiquitous family of multifunctional enzymes that are widely distributed in nature. GSTs have been identified from bacteria, fungi, insects, cephalopods, fish, amphibians, reptiles, avians, mammals and plants. The major role of GSTs is the conjugation of the tripeptide glutathione (GSH; gamma-L-glutamyl-L-cysteinyl-glycine) to a range of electrophilic substances. Ten GSTs from rice, wheat, petunia and Arabidopsis thaliana were overexpressed in Escherichia coli and purified using an Orange A agarose column, S-hexyl glutathione sepharose column, or a nickel chelation column. Using a variety of crystallization screens, crystals were grown for six of the proteins. The X-ray diffraction data collected for two of these proteins enabled their structure to be solved. The first structure to be solved was that of OsGSTU1-1 in complex with GSH, which is a different subgroup of Tau class GSTs from that of the previously reported TaGSTU4-4. OsGSTUI is similar in sequence to the well characterized ZmGSTUI and ZmGSTU2, safener induced enzymes from maize highly active against the diphenyl ether herbicide fluorodifen. In order to determine the structural basis of OsGSTU1 substrate specificity a number of GSH conjugates were prepared. A conjugate of CDNB with GSH was prepared by enzymatic routes using the enzyme ZmGSTF1. The reaction product of the herbicide fluorodifen with GSH was prepared by a two-step synthetic route by preparing a sulfonamide of the phenyl ring and reacting this with GSH. The structure of OsGSTU1-1 was solved with both of these conjugates bound as well as with that of a conjugate of the chloracetanilide herbicide metolachlor with GSH. The structure with metolachlor-GSH bound was useful in structurally characterizing the mode of binding of chloracetanilide herbicides. The structures with the CDNB conjugate with GSH and the reaction product of fluorodifen with GSH were less useful in determining the mode of binding of diphenyl ether herbicides. Both ligands were similar to each other and were found to be sitting between the two lobes of the active site with the nitro group facing inwards. This suggests that the orientation of the molecule within the active site predicted by Dixon et al., 2003 may be incorrect. The structure of OsGSTU4-4 has also been solved in complex with GSH. This enzyme is similar to TaGSTU4-4 and is highly expressed in rice under a variety of stress conditions. A comparison of the three Tau class GST structures now available, along with enzyme assays against a range of xenobiotics substrates has helped to partially understand the activity of Tau class GSTs in herbicide detoxification. The H-site of Tau class GSTs has two lobes. The previously reported structure of TaGSTU4-4 in complex with S-hexylglutathione has the hexyl chain sitting in lobe 'A', but in the structure with metolachlor-GSH the ligand is sitting in lobe 'B' with the gatekeeper Tyr 116 swung away from the active site and disordered. Conformational changes in plant GSTs were also investigated. A comparison of the structures of apo-form ZmGSTF1-1 with the structures of the enzyme in complex with herbicides showed that a conformation change seemed to occur upon ligand binding. Using UV Difference spectroscopy and circular dichroism a change in the conformation of ZmGSTF1-l was seen upon binding of GSH. However, there were problems with the reproducibility of these results. In the case of AtGSTT1-1, which shows a large difference in conformations between molecules in the apo-form of the crystal structure, both UV difference spectroscopy and circular dichroism showed no spectroscopic changes upon GSH binding

The PHD Finger of Human UHRF1 Reveals a New Subgroup of Unmethylated Histone H3 Tail Readers

The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold

Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box). Phylogenetic analyses suggest that this dimerization asymmetry is an ancestral feature necessary for establishing a strong overall dimerization interface, which was progressively modified in other SRs in the course of evolution.journal articl

A structural signature motif enlightens the origin and diversification of nuclear receptors

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the alpha-helix, called pi-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the pi-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the pi-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the pi-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the pi-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the pi-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins

E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. We solved the crystal structures of bovine (BPV1) and human (HPV16) papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

Crystal Structure of Staphopain C from Staphylococcus aureus

International audienceStaphylococcus aureus is a common opportunistic pathogen of humans and livestock that causes a wide variety of infections. The success of S. aureus as a pathogen depends on the production of an array of virulence factors including cysteine proteases (staphopains)—major secreted proteases of certain strains of the bacterium. Here, we report the three-dimensional structure of staphopain C (ScpA2) of S. aureus, which shows the typical papain-like fold and uncovers a detailed molecular description of the active site. Because the protein is involved in the pathogenesis of a chicken disease, our work provides the foundation for inhibitor design and potential antimicrobial strategies against this pathogen

A revisited version of the apo structure of the ligand-binding domain of the human nuclear receptor retinoic X receptor alpha

The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXRalpha, which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a pi-bulge in helix H7, which was originally built as a regular alpha-helix. The presence of several CHAPS molecules, which are visible for the first time in the electron-density map and which stabilize the H1-H3 loop, which contains helix H2, are also revealed. The apo RXR structure has played an essential role in deciphering the molecular mode of action of NR ligands and is still used in numerous biophysical studies. This refined structure should be used preferentially in the future in interpreting experiments as well as for modelling and structural dynamics studies of the apo RXRalpha LBD