Crystal Structure of the Extracellular Domain of the Human Dendritic Cell Surface Marker CD83.

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, ß-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.

Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses.

UNLABELLED: 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. IMPORTANCE: 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.

Investigation of the dynamics of the viral immediate-early protein 1 in different conformations and oligomerization states.

The viral immediate-early protein 1 (IE1) is crucial for efficient replication of cytomegalovirus (CMV). A recent crystal structure of the IE1 protein from rhesus CMV revealed that the protein exhibits a novel fold and crystallizes in two slightly different dimeric arrangements. Molecular dynamics simulations and energetic analyses performed in this study show that both dimers are stable and allowed us to identify a common set of five residues that appear particularly important for dimer formation. These residues are distributed over the entire dimer interface and do not form a typical hot spot for protein interactions. In addition, the dimer interface of IE1 proved to include a high portion of hydrophilic interactions pointing toward the transient nature of dimer formation. Characterization of monomeric and dimeric IE1 revealed three sequentially discontinuous dynamic domains that exhibit correlated motion within the domain and are simultaneously anti-correlated to the adjacent domains. The hinge motions observed between the dynamic domains increase the shape complementarity to the coiled-coil region of tripartite motif proteins, suggesting that the detected dynamics of IE1 might be physiologically important by enabling a better interaction with its cellular target molecules.

Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions.

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.

Structural basis for the recognition of human cytomegalovirus glycoprotein B by a neutralizing human antibody.

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.

Mutations in herpes simplex virus gD protein affect receptor binding by different molecular mechanisms.

Glycoprotein D (gD) is an essential protein of herpes simplex virus-1 (HSV-1) that targets the structurally unrelated receptors HVEM and nectin-1. Receptor binding of gD is accompanied by intramolecular structural rearrangements including the detachment of the C-terminus or formation of an N-terminal hairpin structure. We have investigated several gD mutations that were reported to affect receptor binding affinity or specificity in order to identify their molecular mode of action. Molecular dynamics simulations and subsequent energetic analyses of the gD-receptor complexes reveal that some mutations (M11A, N15A, L28A, T29A) play a more prominent role for HVEM binding than for nectin-1 binding, thereby conferring specificity to receptor recognition. However, our studies show that mutations can also affect the intramolecular structural rearrangement processes in gD. W294A and Q27A mutations facilitate the detachment of the C-terminus, and Q27A additionally hampers the formation of an intramolecular hairpin in gD that is exclusively established upon HVEM binding. The finding that a Q27A mutation affects multiple steps of the receptor binding process offers a molecular explanation for its enhanced nectin-1 affinity and the pronounced receptor specificity. This study also indicates that an inspection of the gD-receptor interfaces alone may be insufficient for predicting the effect of novel mutations that alter receptor specificity. Instead, such an analysis will additionally require to assess the effect of candidate mutation on the preceding steps of gD activation.