Crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing.

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between numerous conformational states and these motions support peptide sampling. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. Here, we present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show pronounced distortions at the N-terminus of the groove. Long stretches of N-terminal amino acid residues are missing in the electron density maps creating an open-ended groove. Our structures also reveal highly unusual features in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations indicate that the complexes have varying degrees of conformational flexibility in a manner consistent with the structures. We suggest that our structures have captured the remarkable molecular dynamics of MHC I-peptide interaction. The visualization of peptide-dependent conformational motions in MHC I is a major step forward in our conceptual understanding of dynamics in high-affinity peptide selection.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Intracellular Sequestration of the NKG2D Ligand MIC B by Species F Adenovirus.

The enteric human adenoviruses of species F (HAdVs-F), which comprise HAdV-F40 and HAdV-F41, are significant pathogens that cause acute gastroenteritis in children worldwide. The early transcription unit 3 (E3) of HAdVs-F is markedly different from that of all other HAdV species. To date, the E3 proteins unique to HAdVs-F have not been characterized and the mechanism by which HAdVs-F evade immune defenses in the gastrointestinal (GI) tract is poorly understood. Here, we show that HAdV-F41 infection of human intestinal HCT116 cells upregulated the expression of MHC class I-related chain A (MIC A) and MIC B relative to uninfected cells. Our results also showed that, for MIC B, this response did not however result in a significant increase of MIC B on the cell surface. Instead, MIC B was largely sequestered intracellularly. Thus, although HAdV-F41 infection of HCT116 cells upregulated MIC B expression, the ligand remained inside infected cells. A similar observation could not be made for MIC A in these cells. Our preliminary findings represent a novel function of HAdVs-F that may enable these viruses to evade immune surveillance by natural killer (NK) cells in the infected gut, thereby paving the way for the future investigation of their unique E3 proteins.

Immune evasion by adenoviruses: a window into host-virus adaptation.

Human adenoviruses (HAdVs) are widespread pathogens that cause a number of partially overlapping, species-specific infections associated with respiratory, urinary, gastrointestinal, and ocular diseases. The early 3 (E3) region of adenoviruses is highly divergent between different species, and it encodes a multitude of proteins with immunomodulatory functions. The study of genetic diversity in the E3 region offers a unique opportunity to gain insight into how the various HAdVs have evolutionarily adapted in response to the selection pressures exerted by host immune defenses. The objective of this review was to discuss subversion of host antiviral immune responses by HAdVs, with a focus on suppression of MHC class I antigen presentation, as a window into host-HAdV adaptation.

Trimming of MHC Class I Ligands by ERAP Aminopeptidases.

Studies over the last decade on characterization of the major histocompatibility complex (MHC) class I antigen presentation pathway have highlighted the importance of antigen processing, peptide transport, peptide trimming, and peptide selection as key stages for the development of optimal peptide repertoires that are presented by MHC class I molecules to cytotoxic T lymphocytes (CTLs). The study of these stages and how they are regulated, is fundamental for progress in understanding the adaptive immune system. Here we describe an in vitro assay monitoring peptide trimming by the human endoplasmic reticulum amino peptidases 1 (ERAP1) and ERAP2 (ERAPs) as a tool to characterize trimming events and gain a better understanding of the role and function of ERAPs in peptide repertoire development. Specifically, our assay allows for monitoring trimming of free but also of MHC I-bound peptides which may reflect the physiological situation best.

In Vitro Studies of MHC Class I Peptide Loading and Exchange.

In the endoplasmic reticulum (ER), MHC class I molecules associate with several specialized proteins, forming a large macromolecular complex referred to as the "peptide-loading complex" (PLC). In the PLC, antigenic peptides undergo a stringent selection process that determines which antigen becomes part of the repertoire presented by MHC class I molecules. This ensures that the immune system elicits robust CD8+ T-cell responses to viruses and solid tumors. The ability to reconstitute in vitro MHC class I molecules in association with key proteins of the PLC provides a mean for studying at the molecular level how antigenic peptides are selected for presentation to CD8+ T-cells. Here, we describe practical procedures for generating a cell-free system made up of MHC class I molecules and tapasin that can be used for mechanistic studies of peptide loading and exchange.

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions.

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.

ERAP1-ERAP2 dimers trim MHC I-bound precursor peptides; implications for understanding peptide editing.

The processing of MHC class I antigenic precursor peptides by the endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 is an important event in the cell biology of antigen presentation. To date, the molecular context by which the ERAP enzymes trim precursor peptides, and how ERAPs shape peptide repertoires, remain open questions. Using ERAP1 and ERAP2 heterodimers (ERAP1/2), and N-terminally extended model and natural peptides in their free and HLA-B*0801-bound forms, we characterized the mode of action of ERAPs. We provide evidence that ERAP1/2 can trim MHC I-bound precursor peptides to their correct and final lengths, albeit more slowly than the corresponding free precursors. Trimming of MHC I-bound precursors by ERAP1/2 increases the conformational stability of MHC I/peptide complexes. From the data, we propose a molecular mechanistic model of ERAP1/2 as peptide editors. Overall, our study provides new findings on a significant issue of the ERAP-mediated processing pathway of MHC class I antigens.

Structure of the Adenovirus Type 4 (Species E) E3-19K/HLA-A2 Complex Reveals Species-Specific Features in MHC Class I Recognition.

Adenoviruses (Ads) subvert MHC class I Ag presentation and impair host anti-Ad cellular activities. Specifically, the Ad-encoded E3-19K immunomodulatory protein targets MHC class I molecules for retention within the endoplasmic reticulum of infected cells. We report the x-ray crystal structure of the Ad type 4 (Ad4) E3-19K of species E bound to HLA-A2 at 2.64-Å resolution. Structural analysis shows that Ad4 E3-19K adopts a tertiary fold that is shared only with Ad2 E3-19K of species C. A comparative analysis of the Ad4 E3-19K/HLA-A2 structure with our x-ray structure of Ad2 E3-19K/HLA-A2 identifies species-specific features in HLA-A2 recognition. Our analysis also reveals common binding characteristics that explain the promiscuous, and yet high-affinity, association of E3-19K proteins with HLA-A and HLA-B molecules. We also provide structural insights into why E3-19K proteins do not associate with HLA-C molecules. Overall, our study provides new information about how E3-19K proteins selectively engage with MHC class I to abrogate Ag presentation and counteract activation of CD8(+) T cells. The significance of MHC class I Ag presentation for controlling viral infections, as well as the threats of viral infections in immunocompromised patients, underline our efforts to characterize viral immunoevasins, such as E3-19K.

Studying MHC class I peptide loading and exchange in vitro.

In the endoplasmic reticulum (ER), MHC class I molecules associate with several specialized proteins, forming a large macromolecular complex referred to as the "peptide-loading complex" (PLC). In the PLC, antigenic peptides undergo a stringent selection process for binding onto MHC class I molecules. This ensures that the immune system elicits robust CD8+ T-cell responses to viruses and solid tumors. The ability to reconstitute in vitro MHC class I molecules in association with key proteins of the PLC provides a mean for studying at the molecular level how antigenic peptides are selected for presentation to CD8+ T-cells. Here, we describe practical procedures for generating a cell-free system involving MHC class I molecules and tapasin, a critical protein of the PLC, that can be used as a versatile tool for biochemical and mechanistic studies of peptide loading and exchange.

Crystal structure of adenovirus E3-19K bound to HLA-A2 reveals mechanism for immunomodulation.

E3-19K binds to and retains MHC class I molecules in the endoplasmic reticulum, suppressing anti-adenovirus activities of T cells. We determined the structure of the adenovirus serotype 2 (Ad2, species C) E3-19K-HLA-A2 complex to 1.95-Å resolution. Ad2 E3-19K binds to the N terminus of the HLA-A2 groove, contacting the α1, α2 and α3 domains and ß(2)m. Ad2 E3-19K has a unique structure comprising a large N-terminal domain, formed by two partially overlapping ß-sheets arranged in a V shape, and a C-terminal α-helix and tail. The structure reveals determinants in E3-19K and HLA-A2 that are important for complex formation; conservation of some of these determinants in E3-19K proteins of different species and MHC I molecules of different loci suggests a universal binding mode for all E3-19K proteins. Our structure is important for understanding the immunomodulatory function of E3-19K.

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules.

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.

Determinants of the endoplasmic reticulum (ER) lumenal-domain of the adenovirus serotype 2 E3-19K protein for association with and ER-retention of major histocompatibility complex class I molecules.

The E3-19K immunomodulatory protein from adenoviruses (Ads) inhibits antigen presentation by major histocompatibility complex (MHC) class I molecules. As a result, the ability of Ad-specific cytotoxic T lymphocytes (CTLs) to lyse infected cells is suppressed. The ER-lumenal domain of E3-19K is subdivided into a variable (residues 1 to ∼78/81) and conserved (residues ∼79/82 to 98) region followed by a linker (residues 99-107). Using molecular and cellular approaches, we characterized in detail the properties of the ER-lumenal domain of E3-19K that enable it to target MHC class I molecules. Proteolysis of recombinant serotype 2 E3-19K (residues 1-100) (with six His residues) generated a large N-terminal (residues 1-88) and a small C-terminal fragment (residues 94-100) in solution. Neither of these fragments associates with HLA-A*1101 as shown by a native gel band-shift assay. In contrast, the N-terminal 1-93 residues of Ad2 E3-19K exhibited the same binding affinity to HLA-A*1101 as E3-19K. Using a site-directed mutational analysis and flow cytometry, we show that Tyr(93), but not Tyr(88), critically modulates the cell-surface expression of MHC class I molecules. Taken together, these results indicate that the sequence comprising residues 89-93 (M(89)SKQY(93)), and in particular Tyr(93), in the conserved region of E3-19K is critical for its immunomodulatory function. Residues 89-93 likely form a linker or loop in E3-19K. Overall, our data provide novel insights into the structure of E3-19K and identify key determinants for association with and ER-retention of its cellular target protein. This knowledge is important for our understanding of the molecular basis of Ad pathogenesis.

Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection.

We examined interactions in a soluble tapasin (TPN)/HLA-B*0801 complex to gain mechanistic insights into the functions of TPN. Results show that TPN acts as a chaperone by increasing the ratio of active-to-inactive peptide-deficient HLA-B*0801 molecules in solution. TPN causes peptides to associate and dissociate faster owing to its effect on widening the binding groove of HLA-B*0801 molecules. Our data indicate that a TPN-assisted mechanism of peptide selection relies on disruption of conserved hydrogen bonds at the C-terminal end of the groove. Peptide sequence-dependent interactions along the entire length of the groove also play a role in this mechanism. We suggest that TPN influences presentation of antigenic peptides according to a mechanistically complicated process in which bound candidate peptides that are unable to conformationally disengage TPN from class I molecules are excluded from the repertoire. Overall, these studies unify our understanding of the functions of TPN.

Allele- and locus-specific recognition of class I MHC molecules by the immunomodulatory E3-19K protein from adenovirus.

The E3-19K protein from human adenoviruses (Ads) retains class I MHC molecules in the endoplasmic reticulum. As a consequence, the cell surface expression of class I molecules is suppressed, allowing Ads to evade immune surveillance. Using native gel electrophoresis, gel filtration chromatography, and surface plasmon resonance, we show that a soluble form of the Ad type 2 (Ad2) E3-19K protein associates with HLA-A and -B molecules; equilibrium dissociation constants were in the nanomolar range and approximately 2.5-fold higher affinity for HLA-A (-A*0201, -A*0301, -A*1101, -A*3301, and -Aw*6801) relative to HLA-B (-B*0702 and -B*0801) molecules. Among the alleles of the HLA-A locus examined, HLA-A*3101 associated approximately 15-fold less avidly with soluble E3-19K. Soluble E3-19K interacted only very weakly with HLA-Cw*0304, and no interaction with HLA-Cw*0401 could be detected under identical conditions. Site-directed mutagenesis and flow cytometry demonstrated that MHC residue 56 plays a critical role in the association and endoplasmic reticulum retention of HLA-A molecules by E3-19K. This delineates the spatial environment around residue 56 as a putative E3-19K interaction surface on class I molecules. Overall, our data imply that a link may exist between host genetic factors and the susceptibility of individuals to Ad infections.

The endoplasmic reticulum lumenal domain of the adenovirus type 2 E3-19K protein binds to peptide-filled and peptide-deficient HLA-A*1101 molecules.

E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A*1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A*1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 +/- 10 nM for peptide-filled molecules and of about 10 microM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A*1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A*1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.

Crystallization and preliminary X-ray crystallographic studies of HLA-A*1101 complexed with an HIV-1 decapeptide.

A major goal of vaccine research for the prevention of AIDS is to determine the immune correlates of protection against HIV-1 infection. In this context, it is of interest to understand how HLA-A*1101, a significantly more prevalent class I allele in a cohort of highly HIV-1-exposed persistently seronegative individuals, functions in relation to protective immunity to HIV-1. Towards this goal, a soluble recombinant HLA-A*1101 molecule has been expressed and used to assemble a complex with beta2-microglobulin and a Nef decapeptide. The HLA-A*1101/beta2m/Nef complex was crystallized by the hanging-drop vapor-diffusion method. The crystal formed in the monoclinic space group P2(1), with unit-cell parameters a = 77.2, b = 88.5, c = 64.8 A, beta = 90.1 degrees, and contains two molecules in the asymmetric unit. A data set to 2.2 A resolution was collected and structure determination by molecular replacement is currently in progress. Understanding the three-dimensional structure of the HLA-A*1101/beta2m/Nef complex may provide insight into the functional role of this class I allele in relation to protective immunity to HIV-1.