PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family ...of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to mutational escape.
OmpG is a general diffusion pore in the E. coli outer membrane with a molecular architecture comprising a 14-stranded β-barrel scaffold and unique structural features. In contrast to other ...non-specific porins, OmpG lacks a central constriction zone and has an exceptionally wide pore diameter of about 13 Å. The equatorial plane of OmpG harbors an annulus of four alternating basic and acidic patches whose function is only poorly characterized. We have investigated the role of charge distribution for ion selectivity and sugar transport with the help of OmpG variants mutated in the annulus. Substituting the glutamate residues of the annulus for histidines or alanines led to a strong reduction in cation selectivity. Replacement of the glutamates in the annulus by histidine residues also disfavored the passage of pentoses and hexoses relative to disaccharides. Our results demonstrate that despite the wide pore diameter, an annulus only consisting of two opposing basic patches confers reduced cation and monosaccharide transport compared to OmpG wild type. Furthermore, randomization of charged residues in the annulus had the potential to abolish pH-dependency of sugar transport. Our results indicate that E15, E31, R92, R111 and R211 in the annulus form electrostatic interactions with R228, E229 and D232 in loop L6 that influence pH-dependency of sugar transport.
Display omitted
•we investigated how charge distribution in the lumen of the general diffusion porin OmpG influences substrate specificity•cation selectivity of OmpG was reduced when luminal glutamate clusters were substituted by histidines or alanines•substitutions of glutamates by histidines improved disaccharide transport relative to monosaccharide transport•alanine substitutions had the opposite effect•residues in the lumen were identified that might confer pH-dependency of OmpG via direct interaction with adjacent residues in loop L6
The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 ...repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.
We use analytical, numerical and experimental methods to characterize the laminar flow inside a Tesla turbine rotor gap. A comparison based on one particular set of operating conditions mutually ...validates the three approaches. The simplicity of the analytical flow model allows for a cost efficient optimization of the underlying turbine parameters. Performance charts exhibit general trends and serve as a guide for preliminary turbine design and optimization. In terms of the ratio of half the gap width to inlet radius and the ratio of outletto inlet radius, the designer of a tesla turbine has to find a compromise between optimal efficiency and optimal power output.
PML is the organizer of cellular structures termed nuclear domain 10 (ND10) or PML-nuclear bodies (PML-NBs) that act as key mediators of intrinsic immunity against human cytomegalovirus (HCMV) and ...other viruses. The antiviral function of ND10 is antagonized by viral regulatory proteins such as the immediate early protein IE1 of HCMV. IE1 interacts with PML through its globular core domain (IE1CORE) and induces ND10 disruption in order to initiate lytic HCMV infection. Here, we investigate the consequences of a point mutation (L174P) in IE1CORE, which was shown to abrogate the interaction with PML, for lytic HCMV infection. We found that a recombinant HCMV encoding IE1-L174P displays a severe growth defect similar to that of an IE1 deletion virus. Bioinformatic modeling based on the crystal structure of IE1CORE suggested that insertion of proline into the highly alpha-helical domain severely affects its structural integrity. Consistently, L174P mutation abrogates the functionality of IE1CORE and results in degradation of the IE1 protein during infection. In addition, our data provide evidence that IE1CORE as expressed by a recombinant HCMV encoding IE1 1-382 not only is required to antagonize PML-mediated intrinsic immunity but also affects a recently described function of PML in innate immune signaling. We demonstrate a coregulatory role of PML in type I and type II interferon-induced gene expression and provide evidence that upregulation of interferon-induced genes is inhibited by IE1CORE. In conclusion, our data suggest that targeting PML by viral regulatory proteins represents a strategy to antagonize both intrinsic and innate immune mechanisms.
PML nuclear bodies (PML-NBs), which represent nuclear multiprotein complexes consisting of PML and additional proteins, represent important cellular structures that mediate intrinsic resistance against many viruses, including human cytomegalovirus (HCMV). During HCMV infection, the major immediate early protein IE1 binds to PML via a central globular domain (IE1CORE), and we have shown previously that this is sufficient to antagonize intrinsic immunity. Here, we demonstrate that modification of PML by IE1CORE not only abrogates intrinsic defense mechanisms but also attenuates the interferon response during infection. Our data show that PML plays a novel coregulatory role in type I as well as type II interferon-induced gene expression, which is antagonized by IE1CORE. Importantly, our finding supports the view that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to inhibit both intrinsic and innate immune defense mechanisms.
In humans, Salmonella enterica infections are responsible for a plethora of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and ...polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secretes SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Secondary structure content and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure contents were in good agreement with estimates derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 Å resolution.
•Soluble domains of SiiD, SiiE and SiiF from Salmonella were produced in high yield.•Circular dichroism spectroscopy indicated the folding of purified proteins.•A SiiD variant comprising the lipoyl and the coiled-coil domain was crystallized.
Cytomegalovirus immediate‐early 1 (IE1) protein is a key viral effector protein that reprograms host cells. Controlled dehydration experiments with IE1 crystals not only extended their diffraction ...limit from 2.85 to 2.3 Å resolution but also triggered a monoclinic to tetragonal space‐group transition with only minor alterations in the unit‐cell parameters. An analysis of the pre‐dehydration and post‐dehydration crystal structures shows how dehydration rearranges the packing of IE1 molecules to meet the unit‐cell constraints of the higher lattice symmetry. The transition from P21 to P43 reduces the number of copies in the asymmetric unit from four to two, and molecules previously related by noncrystallographic symmetry merge into identical crystallographic copies in the tetragonal space group. At the same time, dehydration considerably alters the tertiary structure of one of the two remaining IE1 chains in the asymmetric unit. It appears that this conformational switch is required to compensate for a transition that is assumed to be unfavourable, namely from a highly preferred to a rarely observed space group. At the same time, the dehydration‐triggered molecular reshaping could reveal an inherent molecular flexibility that possibly informs on the biological function of IE1, namely on its binding to target proteins from the host cell.
The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 ...repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca.sup.2+ -binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca.sup.2+ -binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca.sup.2+ -binding sites. BIg domains of SiiE contain distinct Ca.sup.2+ -binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca.sup.2+ -binding sites in SiiE, as well as the importance of Ca.sup.2+ -binding sites in various positions of SiiE. Type I Ca.sup.2+ -binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.
CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound, full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), ...consisting of the extracellular domain only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts its function on dendritic cells and additional immune cells. Here, we report the crystal structure of human sCD83 up to a resolution of 1.7Å solved in three different crystal forms. Interestingly, β-strands C′, C″, and D that are typical for V-set Ig-domains could not be traced in sCD83. Mass spectrometry analyses, limited proteolysis experiments, and bioinformatics studies show that the corresponding segment displays enhanced main-chain accessibility, extraordinary low sequence conservation, and a predicted high disorder propensity. Chimeric proteins with amino acid swaps in this segment show unaltered immune-suppressive activities in a TNF-α assay when compared to wild-type sCD83. This strongly indicates that this segment does not participate in the biological activity of CD83. The crystal structure of CD83 shows the recurrent formation of dimers and trimers in the various crystal forms and reveals strong structural similarities between sCD83 and B7 family members and CD48, a signaling lymphocyte activation molecule family member. This suggests that CD83 exerts its immunological activity by mixed homotypic and heterotypic interactions as typically observed for proteins present in the immunological synapse.
Display omitted
•CD83 is present on many immune cells and exerts immune co-stimulatory activities.•The structure reveals that its surface domain consists of an Ig-like domain only.•β-strands C′, C″, and D, typical for V-set Ig-domains, cannot be traced in CD83.•Sequence swaps show that the absent segment is dispensable for CD83 activity.•Our data indicate that CD83 is active via homotypic and heterotypic interactions.
PDF
