Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD.

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP

Solution structure of the cytochrome P450 reductase–cytochrome c complex determined by neutron scattering

Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved. The function of these complexes requires specificity of the interaction to allow for selective electron transfer but also a fast turnover of the complex, and they are therefore often transient in nature, making them challenging to study. Here, using small-angle neutron scattering with contrast matching with deuterated protein, we report the solution structure of the electron transfer complex between cytochrome P450 reductase (CPR) and its electron transfer partner cytochrome c This is the first reported solution structure of a complex between CPR and an electron transfer partner. The structure shows that the interprotein interface includes residues from both the FMN- and FAD-binding domains of CPR. In addition, the FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of CPR. In summary, our results reveal key details of the CPR catalytic mechanism, including interactions of two domains of the reductase with cytochrome c and motions of these domains relative to one another. These findings shed light on interprotein electron transfer in this system and illustrate a powerful approach for studying solution structures of protein-protein complexes

Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme

Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase

Depression, Somatization and Anxiety in Female Patients with Temporomandibular Disorders (TMD)

The aim of this research was to determine the possible differences in degrees of depression, somatization and anxiety between the acute and chronic female patients with temporomandibular disorders (TMD), and whether these differences exist in healthy female patients. Ninety female patients were involved in this research; 60 of them were TMD patients of the Dental Polyclinic, while other 30 females came for a rutine recall visit and had no problem related to TMD. Patients were aged 22 to 67 years, the average age being 38.5±12 years. All patients were asked to fill in the RDC/TMD protocol and three psychological tests (Emotions Profile Index, Somatization Scale and life Events Scale). Following the analysis of the RDC/TMD protocol and psychological tests, it was determined that the chronic female patients had higher depression and somatization scores in comparison with the acute patients (p<0.01); the acute patients self-perceive higher levels of anxiety in relation to the control group; furthermore, the patients reporting higher levels of depression were more inclined to somatization and had experienced a greater number of stress events in the past six months. It is beyond doubt that patients suffering from the TMD’s exhibit higher levels of depression, somatization and anxiety compared to the healthy ones, which proves that physiological factors may play a predisposing role in combination with reduced level of body tolerance to pain, and a decreased tolerance to stress

Structural basis of STAT2 recognition by IRF9 reveals molecular insights into ISGF3 function

Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9-IAD alone and in a complex with STAT2-CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9-IAD and STAT2-CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells

Crystal Structure and Mechanism of Activation of TANK-Binding Kinase 1

Tank-binding kinase I (TBK1) plays a key role in the innate immune system by integrating signals from pattern-recognition receptors. Here, we report the X-ray crystal structures of inhibitor-bound inactive and active TBK1 determined to 2.6 Å and 4.0 Å resolution, respectively. The structures reveal a compact dimer made up of trimodular subunits containing an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and an α-helical scaffold dimerization domain (SDD). Activation rearranges the KD into an active conformation while maintaining the overall dimer conformation. Low-resolution SAXS studies reveal that the missing C-terminal domain (CTD) extends away from the main body of the kinase dimer. Mutants that interfere with TBK1 dimerization show significantly reduced trans-autophosphorylation but retain the ability to bind adaptor proteins through the CTD. Our results provide detailed insights into the architecture of TBK1 and the molecular mechanism of activation

α-Synuclein Interaction with Lipid Bilayer Discs

α-Synuclein (aSyn) is a 140 residue long protein present in presynaptic termini of nerve cells. The protein is associated with Parkinson's disease, in which case it has been found to self-Assemble into long amyloid fibrils forming intracellular inclusions that are also rich in lipids. Furthermore, its synaptic function is proposed to involve interaction with lipid membranes, and hence, it is of interest to understand aSyn-lipid membrane interactions in detail. In this paper we report on the interaction of aSyn with model membranes in the form of lipid bilayer discs. Using a combination of cryogenic transmission electron microscopy and small-Angle neutron scattering, we show that circular discs undergo a significant shape transition after the adsorption of aSyn. When aSyn self-Assembles into fibrils, aSyn molecules desorb from the bilayer discs, allowing them to recover to their original shape. Interestingly, the desorption process has an all-or-none character, resulting in a binary coexistence of circular bilayer discs with no adsorbed aSyn and deformed bilayer discs having a maximum amount of adsorbed protein. The observed coexistence is consistent with the recent finding of cooperative aSyn adsorption to anionic lipid bilayers

Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction

Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15