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.

Unusual crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing in antigen presentation.

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. We present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also revealed highly unusual features in MHC I and peptide conformations, and in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations showed that the complexes have varying degrees of flexibility in a manner consistent with the structures. We suggest that our structures represent transient substates explored by MHC I molecules during peptide editing. The visualization of peptide-dependent conformational flexibility in MHC I groove is a major step forward in our conceptual understanding of peptide repertoire development in antigen presentation. Our study also raises questions about the role of the N-terminus of the groove in peptide editing.

SARS-CoV-2 ORF8: One protein, seemingly one structure, and many functions.

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 encodes nine accessory proteins that are involved in host-pathogen interaction. ORF8 is unique among these accessory proteins. SARS-CoV-2 ORF8 shares a surprisingly low amino acid sequence similarity with SARS-COV ORF8 (30%), and it is presumed to have originated from bat. Studies have shown that ORF8 exerts multiple different functions that interfere with host immune responses, including the downregulation of MHC class I molecules. These functions may represent strategies of host immune evasion. The x-ray crystal structure of ORF8 revealed an immunoglobulin-like domain with several distinguishing features. To date, there are numerous unanswered questions about SARS-CoV-2 ORF8 protein and its structure-function relationship that we discuss in this mini-review. A better understanding of how ORF8 interacts with components of the immune system is needed for elucidating COVID-19 pathogenesis and to develop new avenues for the treatment of the disease.

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.

ERAP1 enzyme-mediated trimming and structural analyses of MHC I-bound precursor peptides yield novel insights into antigen processing and presentation.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 critically shape the major histocompatibility complex I (MHC I) immunopeptidome. The ERAPs remove N-terminal residues from antigenic precursor peptides and generate optimal-length peptides (i.e. 8-10-mers) to fit into the MHC class I groove. It is therefore intriguing that MHC class I molecules can present N-terminally extended peptides on the cell surface that can elicit CD8+ T-cell responses. This observation likely reflects gaps in our understanding of how antigens are processed by the ERAP enzymes. To better understand ERAPs' function in antigen processing, here we generated a nested set of N-terminally extended 10-20-mer peptides (RA) n AAKKKYCL covalently bound to the human leukocyte antigen (HLA)-B*0801. We used X-ray crystallography, thermostability assessments, and an ERAP1-trimming assay to characterize these complexes. The X-ray structures determined at 1.40-1.65 Å resolutions revealed that the residue extensions (RA) n unexpectedly protrude out of the A pocket of HLA-B*0801, whereas the AAKKKYCL core of all peptides adopts similar, bound conformations. HLA-B*0801 residue 62 was critical to open the A pocket. We also show that HLA-B*0801 and antigenic precursor peptides form stable complexes. Finally, ERAP1-mediated trimming of the MHC I-bound peptides required a minimal length of 14 amino acids. We propose a mechanistic model explaining how ERAP1-mediated trimming of MHC I-bound peptides in cells can generate peptides of canonical as well as noncanonical lengths that still serve as stable MHC I ligands. Our results provide a framework to better understand how the ERAP enzymes influence the MHC I immunopeptidome.

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.

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.

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.

Structure of Arabidopsis chloroplastic monothiol glutaredoxin AtGRXcp.

Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are conserved across species. Arabidopsis thaliana monothiol glutaredoxin AtGRXcp is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been determined at 2.4 A resolution. AtGRXcp has a glutaredoxin/thioredoxin-like fold with distinct structural features that differ from those of dithiol Grxs. The structure reveals that the putative active-site motif CGFS is well defined and is located on the molecular surface and that a long groove extends to both sides of the catalytic Cys97. Structural comparison and molecular modeling suggest that glutathione can bind in this groove and form extensive interactions with conserved charged residues including Lys89, Arg126 and Asp152. Further comparative studies reveal that a unique loop with five additional residues adjacent to the active-site motif may be a key structural feature of monothiol Grxs and may influence their function. This study provides the first structural information on plant CGFS-type monothiol Grxs, allowing a better understanding of the redox-regulation mechanism mediated by these plant Grxs.

Crystal structures of glycosyltransferase UGT78G1 reveal the molecular basis for glycosylation and deglycosylation of (iso)flavonoids.

The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 A resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides a basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.

Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase.

Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between beta3-2 and beta3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.

Crystallization and preliminary X-ray analysis of allene oxide synthase, cytochrome P450 CYP74A2, from Parthenium argentatum.

Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium argentatum) AOS, CYP74A2, was expressed in Escherichia coli. Protein was purified using affinity chromatography and size exclusion chromatography, and then crystallized. Two different crystal forms were obtained from 0.2 M (NH(4))H(2)PO(4), 50% MPD, 0.1 M Tris, pH 8.5 at 277 K using the hanging-drop vapor-diffusion method. Preliminary X-ray analysis was carried out, and the crystals were found to belong to the tetragonal space group I422 with cell parameters a = b = 126.5, c = 163.9 A, and the monoclinic space group C2 with cell parameters a = 336.5, b = 184.2, c = 159.0 A, beta = 118.6 degrees . Diffraction data were collected to 2.4 A resolution from a tetragonal form of crystal using a home X-ray source.

Crystal structure of Medicago truncatula UGT85H2--insights into the structural basis of a multifunctional (iso)flavonoid glycosyltransferase.

(Iso)flavonoids are a diverse group of plant secondary metabolites with important effects on plant, animal and human health. They exist in various glycosidic forms. Glycosylation, which may determine their bioactivities and functions, is controlled by specific plant uridine diphosphate glycosyltransferases (UGTs). We describe a new multifunctional (iso)flavonoid glycosyltransferase, UGT85H2, from the model legume Medicago truncatula with activity towards a number of phenylpropanoid-derived natural products including the flavonol kaempferol, the isoflavone biochanin A, and the chalcone isoliquiritigenin. The crystal structure of UGT85H2 has been determined at 2.1 A resolution, and reveals distinct structural features that are different from those of other UGTs and related to the enzyme's functions and substrate specificities. Structural and comparative analyses revealed the putative binding sites for the donor and acceptor substrates that are located in a large cleft formed between the two domains of the enzyme, and indicated that Trp360 may undergo a conformational change after sugar donor binding to the enzyme. UGT85H2 has higher specificity for flavonol than for isoflavone. Further substrate docking combined with enzyme activity assay and kinetic analysis provided structural insights into this substrate specificity and preference.

Biochemical and structural impact of natural polymorphism in the HLA-A3 superfamily.

Class I alleles of the HLA-A3 superfamily (-A*0301, -A*1101, -A*3101, -A*3301, and -Aw*6801) share largely overlapping peptide repertoires. Cross-reactive T cell responses between HLA-A3-like molecule/peptide complexes have been demonstrated in vitro and during natural diseases. In spite of this immune relatedness, HLA-A3-like molecules exhibit noticeable differences in their antigen-selecting and -presenting properties. Identifying molecular and structural features responsible for these differences is important for understanding how natural polymorphism leads to functional divergence within the HLA-A3 superfamily. Towards this goal, we used an approach that combines thermal stability data on recombinant, soluble HLA-A3-like molecules complexed with a nonamer and decamer HIV-1 peptide, together with a detailed structural analysis of these HLA-A3-like molecule/peptide complexes based on crystal and molecular model structures. Our studies revealed the importance of residues 9 and 67 for modulating peptide selection within the B pocket; of residue 97 for modulating peptide selection within the F pocket interdependently with the presence (or absence) of a middle, secondary anchor residue; and of residues 70, 73, 97, 152, and 156 for modulating peptide presentation in the central region of the groove that leads to altered antigenic surfaces. Overall, our detailed assessment of the biochemical and structural impact of natural polymorphism within the HLA-A3 superfamily has permitted to understand how HLA-A3-like molecules differ at the level of their primary and secondary anchor pockets causing fine differences in their peptide-selecting and -presenting properties. A better understanding of the molecular immunological properties of HLA-A3-like molecules is significantly important for the rationale design of broad peptide-based vaccines.

A biochemical and structural analysis of genetic diversity within the HLA-A*11 subtype.

The HLA-A*11 subtype includes 17 naturally occurring variants (-A*1101 to -A*1117) distributed among different ethnic groups worldwide. At present, only HLA-A*1101 has been characterized at the molecular, structural, and immunological level. Developing similar knowledge on other HLA-A*11 alleles is highly important for bone marrow and graft transplantation. This is also important to better understand disease linkages within the HLA-A*11 subtype given that HLA-A*11 molecules are associated with resistance to acquisition of HIV-1 infection and various autoimmune diseases. To broaden our understanding of HLA-A*11 molecules, we have determined the impact of natural polymorphism on the peptide-binding properties of several HLA-A*11 molecules: -A*1103, -A*1106, -A*1108, -A*1110, -A*1111, and -A*1114. We used an approach that combines data from thermal stability studies of recombinant, soluble forms of these molecules in complex with HIV-1 peptides, together with a detailed structural analysis of the resulting HLA-A*11 molecule/peptide complexes based on crystal and molecular model structures. Our analysis shows that natural polymorphism within the HLA-A*11 subtype is distributed along the alpha1 and alpha2 helices of the peptide-binding groove, in marked contrast to the pattern of polymorphism in HLA-A*2 and HLA-B*27 subtypes. Natural polymorphism greatly altered the abilities of individual -A*11 molecules to form stable complexes with HIV-1 peptides. In comparison to -A*1101, natural polymorphism altered the peptide-presenting properties of -A*1103, -A*1108, and -A*1114 and has the potential to affect the peptide-selecting properties of -A*1106, -A*1110, and -A*1111 as well. Overall, our findings suggest that HLA-A*11 molecules may stimulate alloreactive CD8+ cytotoxic T-cell responses.

Structures of HLA-A*1101 complexed with immunodominant nonamer and decamer HIV-1 epitopes clearly reveal the presence of a middle, secondary anchor residue.

HLA-A*1101 is one of the most common human class I alleles worldwide. An increased frequency of HLA-A*1101 has been observed in cohorts of female sex workers from Northern Thailand who are highly exposed to HIV-1 and yet have remained persistently seronegative. In view of this apparent association of HLA-A*1101 with resistance to acquisition of HIV-1 infection, and given the importance of eliciting strong CTL responses to control and eliminate HIV-1, we have determined the crystal structure of HLA-A*1101 complexed with two immunodominant HIV-1 CTL epitopes: the nonamer reverse transcriptase(313-321) (AIFQSSMTK) and decamer Nef(73-82) (QVPLRPMTYK) peptides. The structures confirm the presence of primary anchor residues P2-Ile/-Val and P9-/P10-Lys, and also clearly reveal the presence of secondary anchor residues P6-Ser for reverse transcriptase and P7-Met for Nef. The overall backbone conformation of both peptides is defined as two bulges that are separated by a more buried middle residue. In this study, we discuss how this topology may offer functional advantages in the selection and presentation of HIV-1 CTL epitopes by HLA-A*1101. Overall, this structural analysis permits a more accurate definition of the peptide-binding motif of HLA-A*1101, the characterization of its antigenic surface, and the correlation of molecular determinants with resistance to HIV-1 infection. These studies are relevant for the rational design of HLA-A*1101-restricted CTL epitopes with improved binding and immunological properties for the development of HIV-1 vaccines.

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.