Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity.

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N-terminal portion which comprises a canonical RING domain, one or two B-box domains and a coiled-coil region that mediates ligase dimerization. Self-association via the coiled-coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin-loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full-length protein. Our data reveal an unexpected diversity in the self-association mechanism of TRIMs that might be crucial for their biological function.

Does it take two to tango? RING domain self-association and activity in TRIM E3 ubiquitin ligases.

TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein.

Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB.

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3.

The Salmonella-secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.

Dynamic Allostery in PLCγ1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR.

Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.

Structural determinants of TRIM protein function.

Tripartite motif (TRIM) proteins constitute one of the largest subfamilies of Really Interesting New Gene (RING) E3 ubiquitin ligases and contribute to the regulation of numerous cellular activities, including innate immune responses. The conserved TRIM harbours a RING domain that imparts E3 ligase activity to TRIM family proteins, whilst a variable C-terminal region can mediate recognition of substrate proteins. The knowledge of the structure of these multidomain proteins and the functional interplay between their constituent domains is paramount to understanding their cellular roles. To date, available structural information on TRIM proteins is still largely restricted to subdomains of many TRIMs in isolation. Nevertheless, applying a combination of structural, biophysical and biochemical approaches has recently allowed important progress to be made towards providing a better understanding of the molecular features that underlie the function of TRIM family proteins and has uncovered an unexpected diversity in the link between self-association and catalytic activity.

Long-term outcomes following "presumed" total parathyroidectomy for secondary hyperparathyroidism of chronic kidney disease.

AIM: The most efficacious surgical treatment for renal hyperparathyroidism is still subject of research. Considering its low incidence rate of long-term relapse, "presumed" total parathyroidectomy without autotrasplantation (TP) may be indicated for secondary hyperparathyroidism (2HPT) in patients with chronic kidney disease (CKD), not eligible for kidney transplantation. The aim of this study was to analyse the TP long-term results in 2HPT haemodialysis (HD) patients. METHOD: Between January 2004 and October 2009, 25 2HPT HD patients, not eligible for kidney transplantation, underwent TP of at least four parathyroid glands. Clinical status and intact parathyroid hormone (iPTH) serum levels were assessed intraoperatively and during a 36-month follow-up. RESULTS: TP improved the typical clinical symptoms and a significant reduction of iPTH serum levels was achieved in each patient. Aparathyroidism was never observed; in case of severe postoperative hypocalcemia, hypocalcemic seizures were never reported and the long-term recurrence rate was 8%. Only one patient received a kidney transplantation. Postoperative cardiovascular events (hypertension, peripheral artery disease, arrhythmia, coronary or cerebrovascular disease) were observed in 32% of cases and mortality rate was 16%. CONCLUSIONS: Considering its low long-term relapse rate and the absence of postoperative aparathyroidism, TP may still be considered the treatment of choice in patients with aggressive forms of 2HPT or of advanced dialytic vintage, with no access to renal transplantation. In case of postoperative hypoparathyroidism, hypocalcaemia can be effectively managed by medical treatment.

Characterisation of HOIP RBR E3 ligase conformational dynamics using integrative modelling.

Multidomain proteins composed of individual domains connected by flexible linkers pose a challenge for structural studies due to their intrinsic conformational dynamics. Integrated modelling approaches provide a means to characterise protein flexibility by combining experimental measurements with molecular simulations. In this study, we characterise the conformational dynamics of the catalytic RBR domain of the E3 ubiquitin ligase HOIP, which regulates immune and inflammatory signalling pathways. Specifically, we combine small angle X-ray scattering experiments and molecular dynamics simulations to generate weighted conformational ensembles of the HOIP RBR domain using two different approaches based on maximum parsimony and maximum entropy principles. Both methods provide optimised ensembles that are instrumental in rationalising observed differences between SAXS-based solution studies and available crystal structures and highlight the importance of interdomain linker flexibility.

Divergent self-association properties of paralogous proteins TRIM2 and TRIM3 regulate their E3 ligase activity.

Tripartite motif (TRIM) proteins constitute a large family of RING-type E3 ligases that share a conserved domain architecture. TRIM2 and TRIM3 are paralogous class VII TRIM members that are expressed mainly in the brain and regulate different neuronal functions. Here we present a detailed structure-function analysis of TRIM2 and TRIM3, which despite high sequence identity, exhibit markedly different self-association and activity profiles. We show that the isolated RING domain of human TRIM3 is monomeric and inactive, and that this lack of activity is due to a few placental mammal-specific amino acid changes adjacent to the core RING domain that prevent self-association but not E2 recognition. We demonstrate that the activity of human TRIM3 RING can be restored by substitution with the relevant region of human TRIM2 or by hetero-dimerization with human TRIM2, establishing that subtle amino acid changes can profoundly affect TRIM protein activity. Finally, we show that TRIM2 and TRIM3 interact in a cellular context via their filamin and coiled-coil domains, respectively.

Retrospective survey of clinical attention taken to osteoporosis in patients admitted in orthopaedic department for fragility fractures.

Considering that to international level it has put in evidence that often the diagnosis of osteoporosis is underestimated and that diagnostic and therapeutic attention of the same one are often neglected, the authors have assessed the degree of care provided by orthopaedic surgeons about the problem of osteoporosis, considering the medical files of orthopaedics department. Then corrective behaviour were proposed.

Characterisation of class VI TRIM RING domains: linking RING activity to C-terminal domain identity.

TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.

Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases.

RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2~Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2~Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5~Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7~Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism.

H-NS is a part of a thermally controlled mechanism for bacterial gene regulation.

Temperature is a primary environmental stress to which micro-organisms must be able to adapt and respond rapidly. Whereas some bacteria are restricted to specific niches and have limited abilities to survive changes in their environment, others, such as members of the Enterobacteriaceae, can withstand wide fluctuations in temperature. In addition to regulating cellular physiology, pathogenic bacteria use temperature as a cue for activating virulence gene expression. This work confirms that the nucleoid-associated protein H-NS (histone-like nucleoid structuring protein) is an essential component in thermoregulation of Salmonella. On increasing the temperature from 25 to 37 degrees C, more than 200 genes from Salmonella enterica serovar Typhimurium showed H-NS-dependent up-regulation. The thermal activation of gene expression is extremely rapid and change in temperature affects the DNA-binding properties of H-NS. The reduction in gene repression brought about by the increase in temperature is concomitant with a conformational change in the protein, resulting in the decrease in size of high-order oligomers and the appearance of increasing concentrations of discrete dimers of H-NS. The present study addresses one of the key complex mechanisms by which H-NS regulates gene expression.

The Biophysical Characterisation and SAXS Analysis of Human NLRP1 Uncover a New Level of Complexity of NLR Proteins.

NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs.

The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR).

In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.

H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein.

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

Flexible stoichiometry and asymmetry of the PIDDosome core complex by heteronuclear NMR spectroscopy and mass spectrometry.

Homotypic death domain (DD)-DD interactions are important in the assembly of oligomeric signaling complexes such as the PIDDosome that acts as a platform for activation of caspase-2-dependent apoptotic signaling. The structure of the PIDDosome core complex exhibits an asymmetric three-layered arrangement containing five PIDD-DDs in one layer, five RAIDD-DDs in a second layer and an additional two RAIDD-DDs. We addressed complex formation between PIDD-DD and RAIDD-DD in solution using heteronuclear nuclear magnetic resonance (NMR) spectroscopy, nanoflow electrospray ionization mass spectrometry and size-exclusion chromatography with multi-angle light scattering. The DDs assemble into complexes displaying molecular masses in the range 130-158kDa and RAIDD-DD:PIDD-DD stoichiometries of 5:5, 6:5 and 7:5. These data suggest that the crystal structure is representative of only the heaviest species in solution and that two RAIDD-DDs are loosely attached to the 5:5 core. Two-dimensional (1)H,(15)N-NMR experiments exhibited signal loss upon complexation consistent with the formation of high-molecular-weight species. (13)C-Methyl-transverse relaxation optimized spectroscopy measurements of the PIDDosome core exhibit signs of differential line broadening, cross-peak splitting and chemical shift heterogeneity that reflect the presence of non-equivalent sites at interfaces within an asymmetric complex. Experiments using a mutant RAIDD-DD that forms a monodisperse 5:5 complex with PIDD-DD show that the spectroscopic signature derives from the quasi- but non-exact equivalent environments of each DD. Since this characteristic was previously demonstrated for the complex between the DDs of CD95 and FADD, the NMR data for this system are consistent with the formation of a structure homologous to the PIDDosome core.

Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.

Structural and functional integration of the PLCγ interaction domains critical for regulatory mechanisms and signaling deregulation.

Multidomain proteins incorporating interaction domains are central to regulation of cellular processes. The elucidation of structural organization and mechanistic insights into many of these proteins, however, remain challenging due to their inherent flexibility. Here, we describe the organization and function of four interaction domains in PLCγ1 using a combination of structural biology and biochemical approaches. Intramolecular interactions within the regulatory region center on the cSH2 domain, the only domain that also interacts with the PLC-core. In the context of fibroblast growth-factor receptor signaling, the coordinated involvement of nSH2 and cSH2 domains mediates efficient phosphorylation of PLCγ1 resulting in the interruption of an autoinhibitory interface by direct competition and, independently, dissociation of PLCγ1 from the receptor. Further structural insights into the autoinhibitory surfaces provide a framework to interpret gain-of-function mutations in PLCγ isoforms linked to immune disorders and illustrate a distinct mechanism for regulation of PLC activity by common interaction domains.

Evaluating the conformation of recombinant domain I of ß(2)-glycoprotein I and its interaction with human monoclonal antibodies.

Pathogenic antiphospholipid antibodies (aPL) cause the antiphospholipid syndrome (APS) by interacting with domain I (DI) of beta-2-glycoprotein I (ß(2)GPI). The aPL/ß(2)GPI complex then exerts pathogenic effects on target cells. We previously described periplasmic bacterial expression of native and mutated variants of DI, and reported the presence of immunodominant epitopes at positions 8-9 (D8/D9) and position 39 (R39). Mutations at these positions strongly influenced the ability of recombinant DI to bind patient-derived IgG aPL and to inhibit pathogenic effects of these aPL in a mouse model of APS. We now describe an improved cytoplasmic bacterial expression system allowing higher yield of DI. We demonstrate that the nuclear magnetic resonance (NMR) spectra of a (15)N,(13)C-isotope-labelled sample of the recombinant DI protein exhibit properties consistent with the structure of DI in crystal structure of intact ß(2)GPI. Mutations at D8/D9 and R39 had limited impact on the NMR spectrum of DI indicating maintenance of the overall fold of the DI domain. We investigated interactions between five variants of DI and ten monoclonal human IgG antibodies, all derived from the IgG aPL antibody IS4 by sequence manipulation and in vitro expression. Arginine residues at positions 100 and 100g in IS4V(H) CDR3 play a particularly important role in binding to DI, but this is unlikely to be due to electrostatic interactions with negatively charged amino acids on DI. Both the strength of binding to DI and the ability to discriminate different DI variants varies between the different IgG antibodies tested. There was no simple relationship between these binding properties and antibody pathogenicity.