Class II-associated Invariant Chain Peptide Research Articles

Structural insights into human MHC-II association with invariant chain

The loading of processed peptides on to major histocompatibility complex II (MHC-II) molecules for recognition by T cells is vital to cell-mediated adaptive immunity. As part of this process, MHC-II associates with the invariant chain (Ii) during biosynthesis in the endoplasmic reticulum to prevent premature peptide loading and to serve as a scaffold for subsequent proteolytic processing into MHC-II-CLIP. Cryo-electron microscopy structures of full-length Human Leukocyte Antigen-DR (HLA-DR) and HLA-DQ complexes associated with Ii, resolved at 3.0 to 3.1 Å, elucidate the trimeric assembly of the HLA/Ii complex and define atomic-level interactions between HLA, Ii transmembrane domains, loop domains, and class II-associated invariant chain peptides (CLIP). Together with previous structures of MHC-II peptide loading intermediates DO and DM, our findings complete the structural path governing class II antigen presentation.

The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

Biomarkers for Diagnosis of Axial Spondyloarthritis, Disease Activity, Prognosis, and Prediction of Response to Therapy.

There exists a major unmet need for biomarkers that can identify axial spondyloarthritis (axSpA) early after disease onset because of the availability of highly effective therapies. Several recent reports have examined the autoantibody response in patients with axSpA through the use of protein microarrays and protein-protein interactions although diagnostic performance of biomarkers identified to date has been inadequate. An example of such a biomarker is protein phosphatase magnesium-dependent 1A. Antibodies to the human leukocyte antigen class II-associated invariant chain peptide (anti-CD74) are candidate diagnostic biomarkers but sensitivity declines with increasing duration of disease. Metabolomic studies have employed nuclear magnetic resonance (NMR) spectrometry to identify disease-specific metabolites related to fat metabolism and intestinal microbial metabolism. A second major unmet need exists for biomarkers of disease activity that have superiority over standard C-reactive protein assessment and reflect MRI inflammation in the axial spine. Several biomarkers reflecting inflammation (calprotectin), angiogenesis (vasoactive endothelial growth factor), and connective tissue turnover (C2M, C3M, and citrullinated metalloproteinase degraded fragment of vimentin) have recently been shown to reflect disease activity when compared with clinical outcomes but comparisons with MRI inflammation are very limited. With increasing availability of highly effective but costly therapies, a third unmet need is biomarkers that can predict response to therapies with different mechanisms of action and are superior to C-reactive protein. Calprotectin is currently the only candidate. Although there are as yet no proven therapies for preventing progression of disease there is an unmet need for biomarkers of prognosis that are more responsive than radiography. Aside from CRP no consistent candidates have emerged. Future studies will need to be prospective, include consecutive patients presenting with undiagnosed back pain, and use more reliable and objective endpoints such as MRI inflammation. Moreover, it has become evident that targeted biomarker studies have not been successful in identifying clinically useful biomarkers and technologies that can simultaneously assess “multiomic” markers will need to be analyzed for future advances. These include more sophisticated metabolomic profiling and universal metabolome-standard (UMS) methodology, next generation RNA sequencing, and affinity-based quantitative proteomics based on the use of nucleic acid binders such as the aptamer-based SOMAscan assay.

Rapid CLIP dissociation from MHC II promotes an unusual antigen presentation pathway in autoimmunity.

A number of autoimmunity-associated MHC class II proteins interact only weakly with the invariant chain-derived class II-associated invariant chain peptide (CLIP). CLIP dissociates rapidly from I-Ag7 even in the absence of DM, and this property is related to the type 1 diabetes-associated β57 polymorphism. We generated knock-in non-obese diabetic (NOD) mice with a single amino acid change in the CLIP segment of the invariant chain in order to moderately slow CLIP dissociation from I-Ag7 These knock-in mice had a significantly reduced incidence of spontaneous type 1 diabetes and diminished islet infiltration by CD4 T cells, in particular T cells specific for fusion peptides generated by covalent linkage of proteolytic fragments within β cell secretory granules. Rapid CLIP dissociation enhanced the presentation of such extracellular peptides, thus bypassing the conventional MHC class II antigen-processing pathway. Autoimmunity-associated MHC class II polymorphisms therefore not only modify binding of self-peptides, but also alter the biochemistry of peptide acquisition.

Cathepsin S inhibition suppresses autoimmune-triggered inflammatory responses in macrophages

In several types of antigen-presenting cells (APCs), Cathepsin S (CatS) plays a crucial role in the regulation of MHC class II surface expression and consequently influences antigen (Ag) presentation of APCs to CD4+ T cells. During the assembly of MHC class II-Ag peptide complexes, CatS cleaves the invariant chain p10 (Lip10) – a fragment of the MHC class II-associated invariant chain peptide. In this report, we used a selective, high-affinity CatS inhibitor to suppress the proteolytic activity of CatS in lymphoid and myeloid cells. CatS inhibition resulted in a concentration-dependent Lip10 accumulation in B cells from both healthy donors and patients with systemic lupus erythematosus (SLE). Furthermore, CatS inhibition led to a decreased MHC class II expression on B cells, monocytes, and proinflammatory macrophages. In SLE patient-derived peripheral blood mononuclear cells, CatS inhibition led to a suppressed secretion of IL-6, TNFα, and IL-10. In a second step, we tested the effect of CatS inhibition on macrophages being exposed to patient-derived autoantibodies against C1q (anti-C1q) that are known to be associated with severe lupus nephritis. As shown previously, those SLE patient-derived high-affinity anti-C1q bound to immobilized C1q induce a proinflammatory phenotype in macrophages. Using this human in vitro model of autoimmunity, we found that CatS inhibition reduces the inflammatory responses of macrophages as demonstrated by a decreased secretion of proinflammatory cytokines, the downregulation of MHC class II and CD80.In summary, we can show that the used CatS inhibitor is able to block Lip10 degradation in healthy donor- and SLE patient-derived B cells and inhibits the induction of proinflammatory macrophages. Thus, CatS inhibition seems to be a promising future treatment of SLE.

Unraveling the structural basis for the unusually rich association of human leukocyte antigen DQ2.5 with class-II-associated invariant chain peptides

Human leukocyte antigen (HLA)-DQ2.5 (DQA1*05/DQB1*02) is a class-II major histocompatibility complex protein associated with both type 1 diabetes and celiac disease. One unusual feature of DQ2.5 is its high class-II-associated invariant chain peptide (CLIP) content. Moreover, HLA-DQ2.5 preferentially binds the non-canonical CLIP2 over the canonical CLIP1. To better understand the structural basis of HLA-DQ2.5's unusual CLIP association characteristics, better insight into the HLA-DQ2.5·CLIP complex structures is required. To this end, we determined the X-ray crystal structure of the HLA-DQ2.5· CLIP1 and HLA-DQ2.5·CLIP2 complexes at 2.73 and 2.20 Å, respectively. We found that HLA-DQ2.5 has an unusually large P4 pocket and a positively charged peptide-binding groove that together promote preferential binding of CLIP2 over CLIP1. An α9-α22-α24-α31-β86-β90 hydrogen bond network located at the bottom of the peptide-binding groove, spanning from the P1 to P4 pockets, renders the residues in this region relatively immobile. This hydrogen bond network, along with a deletion mutation at α53, may lead to HLA-DM insensitivity in HLA-DQ2.5. A molecular dynamics simulation experiment reported here and recent biochemical studies by others support this hypothesis. The diminished HLA-DM sensitivity is the likely reason for the CLIP-rich phenotype of HLA-DQ2.5.

HLA-DP84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway

Classical antigen processing leads to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. Here we show that, unlike other class II molecules, prevalent HLA-DP molecules with β-chains encoding Gly84 (DP84Gly) constitutively present endogenous peptides. DP84Gly does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region, nor does it present CLIP. However, Ii does facilitate the transport of DP84Gly from the endoplasmic reticulum (ER) to the endosomal/lysosomal pathway by transiently binding DP84Gly via a non-CLIP region(s) in a pH-sensitive manner. Accordingly, like class I, DP84Gly constitutively presents endogenous peptides processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP). Therefore, DP84Gly, found only in common chimpanzees and humans, uniquely uses both class I and II antigen-processing pathways to present peptides derived from intracellular and extracellular sources.

HLA–DM Senses Peptide‐MHC Class II Interactions Throughout the Peptide Binding Groove

The major histocompatibility complex class II (MHCII) antigen‐processing pathway is critical for adaptive immunity as the mechanism through which ligands are generated for recognition by CD4 T‐cells. Within the pathway, human leukocyte antigen DM (HLA‐DM) edits the repertoire of peptides that are presented to CD4 T‐cells by catalyzing multiple rounds of peptide exchange and accelerating peptide loading on MHCII molecules. In endosomes, HLA‐DM first catalyzes the dissociation and exchange of the proteolytically processed Ii derived Class II‐associated invariant chain peptide (CLIP) bound to MHCII molecules through a mechanism that follows Michaelis‐Menten kinetics and involves a transient interaction with the peptide‐MHCII complex. HLA‐DM then continues to catalyze the rate of dissociation and exchange of all other peptides. The catalytic potency of DM varies such that peptide exchange is markedly enhanced on some peptide‐MHCII complexes and not others. This capacity to differentially edit peptide‐MHCII complexes has important biological implications through its effect of skewing the repertoire of foreign and self‐peptide complexes available for activation or tolerance induction of CD4 T‐cells. Despite the large body of evidence indicating the central role of HLA‐DM in the MHCII antigen‐processing pathway, the catalytic mechanism and the rules that govern the susceptibility of peptide‐MHCII complexes to DM mediated editing are not completely understood. Evidence suggests that unstable interactions at the N‐terminus of bound peptides is the key determinant of peptide‐MHCII complex sensitivity to HLA‐DM and that partial dissociation of a peptides N‐terminus is necessary for this sensitivity. Herein, we employed a fluorescence polarization assay to independently assess the effective affinity and catalytic turnover, components of the catalytic mechanism, in real‐time kinetic measurements of DM activity. Using a series of amino acid substituted peptides, we have analyzed the effects of disfavored interactions on the kinetics of DM editing. We find that disruptions throughout the peptide binding groove contribute to the effective binding affinity of DM for a given peptide complex and to the rate of peptide dissociation from the DM–peptide‐MHCII catalytic complex. Experiments with mutant peptide‐MHCII complexes that disrupt elements in the conserved hydrogen bond network show that disruption near the C‐terminus results in increased DM affinity and catalytic turnover. Furthermore, we found that complexes with an N‐terminal truncated form of the immunodominate HA peptide, resulting in the loss of three H‐bonds, showed an unexpected increase in catalytic turnover when compared to the unmodified HA‐MHCII complex. This result was even more pronounced when a conserved bidentate H‐bond forming residue near the P1 anchor of the HA‐MHCII complex was disrupted by site‐directed mutagenesis. Together, this implies that peptide‐MHCII interactions near the N‐terminal segment of a peptide are intact during the functionally active DM‐peptide‐MHCII catalytic complex and that interactions at the DM‐MHCII contact interface are intimately linked to unstable interactions throughout the peptide‐binding groove.Support or Funding InformationSupported in part by NIH grants AI30554 and AI33614

Type 1 diabetes associated HLA‐DQ2 and DQ8 molecules are relatively resistant to HLA‐DM mediated release of invariant chain‐derived CLIP peptides

HLA-DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4(+) T cells. Individuals expressing HLA-DQ2 or DQ8, and DQ2/8 trans-dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM-mediated editing of CLIP was further confirmed by HPLC-MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D-associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D-susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2-CLIP complexes are highly resistant to DM editing, whereas DQ8-CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM-binding region. Our findings show that T1D-susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D-susceptible DQ molecules to DM editing and preferential presentation of T1D-associated autoantigenic peptides may contribute to the pathogenesis of T1D.

Immune Response in Acute Myeloid Leukemia with Mutated Nucleophosmin

In our recent work, we described changes in HLA class I frequencies in AML patients with nucleophosmin (NPM) mutations. Several allelic groups were found to be depleted in patients with C-terminal NPM mutations (NPMc+), but not in patients with wild-type NPM, and these differences corresponded well to in silico prediction of NPM-derived peptides binding to HLA class I. Moreover, patients with "favorable" HLA type (i.e., expressing HLA alleles suitable for presentation of NPM-derived peptides) were found to have significantly better overall survival than those without suitable HLA alleles. Altogether, our results suggested that spontaneous anti-NPM immune response may prevent AML development and helps achieve durable cure.In this contribution, an extended patient cohort (N = 71 patients with all available data) was divided into four groups according to HLA type (favorable/unfavorable) and Flt3-ITD status. Patients expressing suitable HLA alleles and not having internal tandem duplications in Flt3 gene (HLA+/Flt3-) had the best overall survival (Figure 1). The impact of unfavorable HLA type alone was comparable to that of Flt3-ITD positivity while the combination of both adverse factors was associated with particularly poor outcome (p = 0.0066 for trend).On the basis of these results, we believe that NPMc+ AML patients with favorable HLA type (about 65% of all NPMc+ AML patients) could benefit from an immunomodulatory therapy. Besides inhibitory receptors (such as PD-1, CTLA-4, TIM-3), regulatory T-cells or decreased HLA expression, reduced antigen presentation by leukemic cells belongs to possible mechanisms of immune escape. Insufficiency of molecular complexes such as the proteasome or the transport-associated protein (TAP) can result in increased presentation of Class II-associated invariant chain peptide (CLIP). Indeed, increased CLIP expression on residual leukemic blasts has recently been reported to correlate with increased relapse risk in AML. Interestingly, valproic acid used in combination with standard chemotherapy has been described to significantly decrease the relapse rate in elderly AML patients with mutated NPM (but not in other AML subgroups). This could be associated with restored antigen presentation following treatment with histonedeacetylase inhibitors. In our experiments, we observed marked reduction of CLIP expression on primary NPMc+ AML blasts following in vitro treatment with suberoylanilide hydroxamic acid (SAHA, Figure 2). Both the fraction of CLIP-positive cells (open circles) and CLIP surface density on positive cells (closed circles) were reduced. HLA class I expression was normal and was not affected by SAHA treatment. Importantly, the effect on CLIP expression occurred even at subtoxic (0.5 µM) drug concentration. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biomarkers in axial spondyloarthritis.

To assess the literature for biomarker validation studies that address key unmet needs related to the evaluation and management of patients with axial spondyloarthritis (SpA). This review focused on biomarkers facilitating early diagnosis and reflecting disease activity, structural damage on radiography, and clinical response to major therapies. Early diagnosis may be facilitated by measurement of antibodies to the human leukocyte antigen class II-associated invariant chain peptide (anti-CD74) but sensitivity declines with increasing duration of disease. No disease activity biomarkers have demonstrated consistent superiority over standard C-reactive protein (CRP), and future validation should employ multivariate analysis aimed at demonstrating the added value of any associated biomarkers beyond available clinical parameters of disease activity and the use of magnetic resonance imaging inflammation as the primary endpoint. Several biomarkers reflecting inflammation (CRP and calprotectin), angiogenesis (vasoactive endothelial growth factor), and connective tissue turnover (C2 M, C3 M, and citrullinated metalloproteinase degraded fragment of vimentin ) have recently been shown to reflect radiographic progression in multivariate studies adjusted for baseline severity. Future studies should be prospective and demonstrate that predictive capacity adds to the information provided by known predictors such as CRP and baseline modified Stoke AS Spine Score. Calprotectin is a promising predictor of response to major therapies for axial SpA. Several promising biomarkers addressing major unmet clinical needs require further validation in prospective studies.

Class II-associated invariant chain peptide as predictive immune marker in minimal residual disease in acute myeloid leukemia

The majority of patients with acute myeloid leukemia (AML) reach complete remission after high-dose chemotherapy. Still, half of these patients experience a relapse due to presence of minimal residual disease (MRD). Here we discuss the poor prognostic role of class II-associated invariant chain peptide (CLIP) expression on residual leukemic cells.

High class II-associated invariant chain peptide expression on residual leukemic cells is associated with increased relapse risk in acute myeloid leukemia.

The presence of class II-associated invariant chain (CLIP) on leukemic cells is negatively associated with clinical outcome in untreated acute myeloid leukemia (AML). CLIP plays a role in the immune escape of leukemic cells, suggesting that it impairs the immunogenicity of minimal residual disease (MRD) cells causing a relapse. Here, we demonstrate that CLIP expression on leukemia-associated phenotype (LAP)-positive cells during follow-up is significantly correlated with a shortened relapse-free survival, even in those patients who are generally considered as MRD(low) (0.01-0.1% LAP(+) cells). Consequently, CLIP evaluation could be of additional value in the evaluation of MRD to predict a relapse of AML.

N-terminal functional region of the invariant chain efficiently targets the binding of a CTL epitope to MHC class I molecules during cross-presentation.

Cross-presentation (CP) is important for priming T cell responses to many viral, bacterial, and tumor antigens. Here, we designed two Ii mutants, based on evidence that the invariant chain (Ii, also named CD74) binds newly synthesized MHC class I molecules with the class II-associated invariant chain peptide (CLIP) region of Ii, which occupies the peptide-binding groove. Specifically, we designed (1) Ii-O257, which is a CLIP-substituted Ii chimer, in which OVA257-264 (SIINFEKL) was substituted for CLIP, and (2) Ii-, also named CT257, which is a C-terminal truncated form of Ii-O257 that contains the N-terminal flanking region of Ii. We immunized C57BL/6 mice with these recombinant proteins. Real-time PCR detected that mice immunized with either Ii-O257 or Ii-CT257 recombinant proteins exhibited increased IFN-γ mRNA expression (approximately 11-fold and 13-fold, respectively) and increased IL-2 mRNA expression (approximately 9-fold and 11-fold, respectively), compared to mice immunized with the OVA257-264 peptide. In vivo cytokine analysis showed that recombinant Ii proteins were highly efficient at activating T cells. Confocal microscopy and co-immunoprecipitation showed that the 2 Ii-OVA257-264 chimers are associated intracellularly with H-2K(b) molecules. Thus, Ii-CT257 (amino acids 1-89) binds stably to MHC class I with high affinity, indicating that it is a minimal functional fragment of the Ii immune vector. In conclusion, the N-terminal functional region of the Ii fusion protein containing CTL epitopes might prove to be useful for developing peptide or DNA vaccines that use CP as the main mechanism for CD8(+) T cell stimulation.

HLA-DO increases bacterial superantigen binding to human MHC molecules by inhibiting dissociation of class II-associated invariant chain peptides

HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule (MHCII). It forms a stable complex with HLA-DM (H2-M in mice) and shapes the MHC class II-associated peptide repertoire. Here, we tested the impact of HLA-DO and H2-O on the binding of superantigens (SAgs), which has been shown previously to be sensitive to the structural nature of the class II-bound peptides. We found that the binding of staphylococcal enterotoxin (SE) A and B, as well as toxic shock syndrome toxin 1 (TSST-1), was similar on the HLA-DO(+) human B cell lines 721.45 and its HLA-DO(-) counterpart. However, overexpressing HLA-DO in MHC class II(+) HeLa cells (HeLa-CIITA-DO) improved binding of SEA and TSST-1. Accordingly, knocking down HLA-DO expression using specific siRNAs decreased SEA and TSST-1 binding. We tested directly the impact of the class II-associated invariant chain peptide (CLIP), which dissociation from MHC class II molecules is inhibited by overexpressed HLA-DO. Loading of synthetic CLIP on HLA-DR(+) cells increased SEA and TSST-1 binding. Accordingly, knocking down HLA-DM had a similar effect. In mice, H2-O deficiency had no impact on SAgs binding to isolated splenocytes. Altogether, our results demonstrate that the sensitivity of SAgs to the MHCII-associated peptide has physiological basis and that the effect of HLA-DO on SEA and TSST-1 is mediated through the inhibition of CLIP release.

FRI0279 High prevalence of anti-CD 74 antibodies with specificity for the class II-associated invariant chain peptide (clip) in patients with axial spondyloarthritis but not in controls

BackgroundThe pathogenesis of axial spondyloarthritis (axSpA) including ankylosing spondylitis (AS), although known to be strongly associated with genes such as the human leukocyte antigen (HLA) B27, is still largely unclear....

AB0898 Biomarker for spondyloarthritis: Autoantibodies against class II-associated invariant chain peptide (clip)

BackgroundSpondyloarthritis (SpA) is relatively common inflammatory disorder with a frequency of 1-2% in the European population (1). Establishing the diagnosis however may be difficult, since abnormalities in conventional X-ray develop...

Autoantibodies against CD74 in spondyloarthritis

BackgroundSpondyloarthritis (SpA) is a common debilitating inflammatory disorder. Establishing the diagnosis is often difficult, since abnormalities in conventional X-ray develop with a latency of several years and only HLA-B27 is...

High prevalence of anti-CD74 antibodies specific for the HLA class II-associated invariant chain peptide (CLIP) in patients with axial spondyloarthritis

ObjectivesThe pathogenesis of axial spondyloarthritis (axSpA) is still unclear. There is a strong association with HLA-B27 and other genes. Recently, anti-CD74 antibodies with specificity to a class II-associated invariant chain...

X-ray crystal structure of HLA-DQ2 in complex with CLIP1 and CLIP2 (P5026)

Abstract The class II MHC protein HLA-DQ2 is strongly associated with type 1 diabetes and celiac disease. CLIP (MHC class II-associated invariant chain peptide) occupies the peptide binding groove of all newly synthesized class II MHC proteins until HLA-DM replaces it with an antigen peptide. We have determined the atomic structure of HLA-DQ2-CLIP1 and HLA-DQ2-CLIP2 complex at 2.8 Ang and 2.2 Ang, respectively. Our crystal structure shows that MRMATPLLM of CLIP1 occupies the P1 to P9 pockets of HLA-DQ2. For CLIP2, PLLMQALPM occupies the P1 to P9 pockets of HLA-DQ2. We present our analysis of the MHC-peptide interaction in these two structures, and we highlight the structural similarities and differences compared to other class II MHC-CLIP complex structures reported to date.