The interaction between signal regulatory protein alpha (SIRPα) and CD47: structure, function, and therapeutic target.

CD47 is a broadly expressed membrane protein that interacts with the myeloid inhibitory immunoreceptor SIRPα (also termed CD172a or SHPS-1). SIRPα is the prototypic member of the SIRP paired receptor family of closely related SIRP proteins. Engagement of SIRPα by CD47 provides a downregulatory signal that inhibits host cell phagocytosis, and CD47 therefore functions as a "don't-eat-me" signal. Here, we discuss recent structural analysis of CD47-SIRPα interactions and implications of this for the function and evolution of SIRPα and paired receptors in general. Furthermore, we review the proposed roles of CD47-SIRPα interactions in phagocytosis, (auto)immunity, and host defense, as well as its potential significance as a therapeutic target in cancer and inflammation and for improving graft survival in xenotransplantation.

CD225 Proteins: A Family Portrait of Fusion Regulators.

The CD225 superfamily regulates vesicular membrane fusion events essential to neurotransmission, immunity, development, and metabolism. Its importance to physiology is reinforced by the identification of polymorphisms associated with disease. This article highlights the shared features that drive the function of CD225 proteins such as interferon-inducible transmembrane proteins 3 (IFITM3) and proline-rich transmembrane protein 2 (PRRT2) and is intended to catalyze efforts towards characterizing the lesser-known family members.

Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

Biomimetic Design of Peptide Inhibitor to Block CD47/SIRPα Interactions.

CD47 on the surface of tumor cells has become a research hot spot in immunotherapy and anticancer therapy, as it can bind to SIRPα protein on the surface of macrophages, which ultimately leads to immune escape of tumor cells. In the present study, molecular interactions between CD47 and human SIRPα proteins (including variant 1, V1 and variant 2, V2) were analyzed through molecular dynamics (MD) simulation and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Hydrophobic interactions were found as the main driving force for the binding of CD47 on SIRPα. The residues including pyroglutamate acid (Z)1, L2, E35, Y37, E97, L101, and T102 of CD47 were identified with a significant favorable contribution to the binding of CD47 on SIRPα (both V1 and V2). Based on this, a peptide inhibitor library with the sequence ZLXRTLXEXY was designed (X represents the arbitrary residue of 20 standard amino acids) and then screened using molecular docking, MD simulations, and experimental validation. Finally, a peptide ZLIRTLHEWY was determined with high affinity with SIRPα from 8000 candidates, containing 6/10 residues favorable for the binding on SIRPα V1 and 8/10 residues favorable for the binding on SIRPα V2, which was thus considered to have potential anticancer function.

Binding Mechanism of CD47 with SIRPα Variants and Its Antibody: Elucidated by Molecular Dynamics Simulations.

The intricate complex system of the differentiation 47 (CD47) and the signal-regulatory protein alpha (SIRPα) cluster is a crucial target for cancer immunotherapy. Although the conformational state of the CD47-SIRPα complex has been revealed through crystallographic studies, further characterization is needed to fully understand the binding mechanism and to identify the hot spot residues involved. In this study, molecular dynamics (MD) simulations were carried out for the complexes of CD47 with two SIRPα variants (SIRPαv1, SIRPαv2) and the commercially available anti-CD47 monoclonal antibody (B6H12.2). The calculated binding free energy of CD47-B6H12.2 is lower than that of CD47-SIRPαv1 and CD47-SIRPαv2 in all the three simulations, indicating that CD47-B6H12.2 has a higher binding affinity than the other two complexes. Moreover, the dynamical cross-correlation matrix reveals that the CD47 protein shows more correlated motions when it binds to B6H12.2. Significant effects were observed in the energy and structural analyses of the residues (Glu35, Tyr37, Leu101, Thr102, Arg103) in the C strand and FG region of CD47 when it binds to the SIRPα variants. The critical residues (Leu30, Val33, Gln52, Lys53, Thr67, Arg69, Arg95, and Lys96) were identified in SIRPαv1 and SIRPαv2, which surround the distinctive groove regions formed by the B2C, C'D, DE, and FG loops. Moreover, the crucial groove structures of the SIRPα variants shape into obvious druggable sites. The C'D loops on the binding interfaces undergo notable dynamical changes throughout the simulation. For B6H12.2, the residues Tyr32LC, His92LC, Arg96LC, Tyr32HC, Thr52HC, Ser53HC, Ala101HC, and Gly102HC in its initial half of the light and heavy chains exhibit obvious energetic and structural impacts upon binding with CD47. The elucidation of the binding mechanism of SIRPαv1, SIRPαv2, and B6H12.2 with CD47 could provide novel perspectives for the development of inhibitors targeting CD47-SIRPα.

A MD Simulation Prediction for Regulation of N-Terminal Modification on Binding of CD47 to CD172a in a Force-Dependent Manner.

Cancer cells can evade immune surveillance through binding of its transmembrane receptor CD47 to CD172a on myeloid cells. CD47 is recognized as a promising immune checkpoint for cancer immunotherapy inhibiting macrophage phagocytosis. N-terminal post-translated modification (PTM) via glutaminyl cyclase is a landmark event in CD47 function maturation, but the molecular mechanism underlying the mechano-chemical regulation of the modification on CD47/CD172a remains unclear. Here, we performed so-called "ramp-clamp" steered molecular dynamics (SMD) simulations, and found that the N-terminal PTM enhanced interaction of CD172a with CD47 by inducing a dynamics-driven contraction of the binding pocket of the bound CD172a, an additional constraint on CYS15 on CD47 significantly improved the tensile strength of the complex with or without PTM, and a catch bond phenomenon would occur in complex dissociation under tensile force of 25 pN in a PTM-independent manner too. The residues GLN52 and SER66 on CD172a reinforced the H-bonding with their partners on CD47 in responding to PTM, while ARG69 on CD172 with its partner on CD47 might be crucial in the structural stability of the complex. This work might serve as molecular basis for the PTM-induced function improvement of CD47, should be helpful for deeply understanding CD47-relevant immune response and cancer development, and provides a novel insight in developing of new strategies of immunotherapy targeting this molecule interaction.

SIRPα-specific monoclonal antibody enables antibody-dependent phagocytosis of neuroblastoma cells.

Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting. Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6 mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.

Functional Heterogeneity of Mammalian IFITM Proteins against HIV-1.

Interferon-induced transmembrane proteins (IFITMs) are a family of interferon-inducible proteins that inhibit a broad range of viruses by interfering with viral-to-cellular membrane fusion. The antiviral activity of IFITMs is highly regulated by several posttranslational modifications and by a number of protein domains that modulate steady-state protein levels, trafficking, and antiviral effectiveness. Taking advantage of the natural diversity existing among IFITMs of different animal species, we have compared 21 IFITMs for their ability to inhibit HIV-1 at two steps, during virus entry into cells (target cell protection) and during the production of novel virion particles (negative imprinting of virion particles' infectivity). We found a high functional heterogeneity among IFITM homologs with respect to both antiviral modalities, with IFITM members that exhibit enhanced viral inhibition, while others have no ability to block HIV-1. These differences could not be ascribed to known regulatory domains and could only be partially explained through differential protein stability, implying the existence of additional mechanisms. Through the use of chimeras between active and inactive IFITMs, we demonstrate that the cross talk between distinct domains of IFITMs is an important contributor of their antiviral potency. Finally, we identified murine IFITMs as natural variants competent for target cell protection, but not for negative imprinting of virion particles' infectivity, suggesting that the two properties may, at least in principle, be uncoupled. Overall, our results shed new light on the complex relationship between IFITMs and viral infection and point to the cross talk between IFITM domains as a novel layer of regulation of their activity. IMPORTANCE IFITMs are broad viral inhibitors capable of interfering with both early and late phases of the replicative cycle of many different viruses. By comparing 21 IFITM proteins issued from different animal species for their ability to inhibit HIV-1, we have identified several that exhibit either enhanced or impaired antiviral behavior. This functional diversity is not driven by differences in known domains and can only be partly explained through differential protein stability. Chimeras between active and inactive IFITMs point to the cross talk between individual IFITM domains as important for optimal antiviral activity. Finally, we show that murine IFITMs are not capable of decreasing the infectivity of newly produced HIV-1 virion particles, although they retain target cell protection abilities, suggesting that these properties may be, in principle, disconnected. Overall, our results shed new light on the complex layers of regulation of IFITM proteins and enrich our current understanding of these broad antiviral factors.

Crystal Structure of Human CD47 in Complex with Engineered SIRPα.D1(N80A).

Background: Targeting the CD47/SIRPα signaling pathway represents a novel approach to enhance anti-tumor immunity. However, the crystal structure of the CD47/SIRPα has not been fully studied. This study aims to analyze the structure interface of the complex of CD47 and IMM01, a novel recombinant SIRPα-Fc fusion protein. Methods: IMM01-Fab/CD47 complex was crystalized, and diffraction images were collected. The complex structure was determined by molecular replacement using the program PHASER with the CD47-SIRPαv2 structure (PDB code 2JJT) as a search model. The model was manually built using the COOT program and refined using TLS parameters in REFMAC from the CCP4 program suite. Results: Crystallization and structure determination analysis of the interface of IMM01/CD47 structure demonstrated CD47 surface buried by IMM01. Comparison with the literature structure (PDB ID 2JJT) showed that the interactions of IMM01/CD47 structure are the same. All the hydrogen bonds that appear in the literature structure are also present in the IMM01/CD47 structure. These common hydrogen bonds are stable under different crystal packing styles, suggesting that these hydrogen bonds are important for protein binding. In the structure of human CD47 in complex with human SIRPα, except SER66, the amino acids that form hydrogen bonds are all conserved. Furthermore, comparing with the structure of PDB ID 2JJT, the salt bridge interaction from IMM01/CD47 structure are very similar, except the salt bridge bond between LYS53 in IMM01 and GLU106 in CD47, which only occurs between the B and D chains. However, as the side chain conformation of LYS53 in chain A is slightly different, the salt bridge bond is absent between the A and C chains. At this site between chain A and chain C, there are a salt bridge bond between LYS53 (A) and GLU104 (C) and a salt bridge bond between HIS56 (A) and GLU106 (C) instead. According to the sequence alignment results of SIRPα, SIRPß and SIRPγ in the literature of PDB ID 2JJT, except ASP100, the amino acids that form common salt bridge bonds are all conserved. Conclusion: Our data demonstrated crystal structure of the IMM01/CD47 complex and provides a structural basis for the structural binding interface and future clinical applications.

Selection and Characterization of FD164, a High-Affinity Signal Regulatory Protein α Variant with Balanced Safety and Effectiveness, from a Targeted Epitope Mammalian Cell-Displayed Antibody Library.

Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.

Enhancement of epidermal growth factor receptor antibody tumor immunotherapy by glutaminyl cyclase inhibition to interfere with CD47/signal regulatory protein alpha interactions.

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.

Design, Synthesis and Evaluation of Novel Derivatives of Curcuminoids with Cytotoxicity.

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,ß-unsaturated ß-diketone, α,ß-unsaturated ketone and ß'-hydroxy-α,ß-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC50 values of all the synthesized derivatives, most α,ß-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas ß'-hydroxy-α,ß-unsaturated ketones and α,ß-unsaturated ß-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,ß-unsaturated ketone complex for help in drug design.

Interferon-induced transmembrane proteins inhibit cell fusion mediated by trophoblast syncytins.

Type I interferon (IFN) induced by virus infections during pregnancy can cause placental damage, but the mechanisms and identities of IFN-stimulated genes that are involved in this damage remain under investigation. The IFN-induced transmembrane proteins (IFITMs) inhibit virus infections by preventing virus membrane fusion with cells and by inhibiting fusion of infected cells (syncytialization). Fusion of placental trophoblasts via expression of endogenous retroviral fusogens known as syncytins forms the syncytiotrophoblast, a multinucleated cell structure essential for fetal development. We found here that IFN blocks fusion of BeWo human placental trophoblasts. Stably expressed IFITM1, -2, and -3 also blocked fusion of these trophoblasts while making them more resistant to virus infections. Conversely, stable IFITM knockdowns in BeWo trophoblasts increased their spontaneous fusion and allowed fusion in the presence of IFN while also making the cells more susceptible to virus infection. We additionally found that exogenous expression of IFITMs in HEK293T cells blocked fusion with cells expressing syncytin-1 or syncytin-2, confirming the ability of IFITMs to block individual syncytin-mediated fusion. Overall, our data indicate that IFITMs inhibit trophoblast fusion and suggest that there may be a critical balance between these antifusogenic effects and the beneficial antiviral effects of IFITMs in virus infections during pregnancy.

BC2L-C N-Terminal Lectin Domain Complexed with Histo Blood Group Oligosaccharides Provides New Structural Information.

Lectins mediate adhesion of pathogens to host tissues, filling in a key role in the first steps of infection. Belonging to the opportunistic pathogen Burkholderia cenocepacia, BC2L-C is a superlectin with dual carbohydrate specificity, believed to mediate cross-linking between bacteria and host cells. Its C-terminal domain binds to bacterial mannosides while its N-terminal domain (BCL2-CN) recognizes fucosylated human epitopes. BC2L-CN presents a tumor necrosis factor alpha (TNF-) fold previously unseen in lectins with a novel fucose binding mode. We report, here, the production of a novel recombinant form of BC2L-CN (rBC2L-CN2), which allowed better protein stability and unprecedented co-crystallization with oligosaccharides. Isothermal calorimetry measurements showed no detrimental effect on ligand binding and data were obtained on the binding of Globo H hexasaccharide and l-galactose. Crystal structures of rBC2L-CN2 were solved in complex with two blood group antigens: H-type 1 and H-type 3 (Globo H) by X-ray crystallography. They provide new structural information on the binding site, of importance for the structural-based design of glycodrugs as new antimicrobials with antiadhesive properties.

The counterbalance theory for evolution and function of paired receptors.

Paired receptors are families of membrane proteins containing similar extracellular regions but differing in their potential for signaling with one type able to give inhibitory signals and the other activating. Inhibitory receptors could be good targets for pathogens to restrict immune responses against them. Here we suggest that activating members may have evolved to counterbalance pathogens utilizing the inhibitory pathway. Thus, if a pathogen utilizes any part of the inhibitory receptor to downregulate responses against itself, it may, because of similarities in structure, also bind the activating receptor and give an opposing signal. We evaluate recent structural data on SIRPalpha (signal regulatory protein) and LILRB1 (leukocyte immunoglobulin-like receptor subfamily B member 1) showing evidence of pathogen pressure in nonligand-binding regions of these receptors together with data on pathogen binding to PIRs (paired Ig-like receptors) to provide support for this theory.

Polymorphisms in the human inhibitory signal-regulatory protein α do not affect binding to its ligand CD47.

CD47 is a widely distributed membrane protein that interacts with signal-regulatory protein α (SIRPα), an inhibitory receptor on myeloid cells that gives a "don't-eat-me" signal. Manipulation of the interaction is of considerable interest in the immunotherapy of cancer and in xenotransplantation. The amino-terminal ligand binding domain of SIRPα is highly polymorphic in contrast to the single Ig-like domain of CD47. There is confusion as to whether the polymorphisms will affect ligand binding, but this is an important point for this interaction and other paired receptors being considered as targets for therapy. We show by x-ray crystallography that one human SIRPα allele differing in 13 amino acid residues has a very similar binding site and that several different alleles all bind CD47 with similar affinity as expected because the residues are mostly surface-exposed and distant from the binding site. A peptide from the binding site of CD47 has been reported to mimic the CD47 interaction with SIRPα, but we could find no binding. We discuss the possible pitfalls in determining the affinity of weak interactions and also speculate on how SIRPα polymorphisms may have been selected by pathogens and how this may also be true in other paired receptors such as the KIRs.

Palmitoylation on conserved and nonconserved cysteines of murine IFITM1 regulates its stability and anti-influenza A virus activity.

The interferon-induced transmembrane proteins (IFITMs) restrict infection by numerous viruses, yet the importance and regulation of individual isoforms remains unclear. Here, we report that murine IFITM1 (mIFITM1) is palmitoylated on one nonconserved cysteine and three conserved cysteines that are required for anti-influenza A virus activity. Additionally, palmitoylation of mIFITM1 regulates protein stability by preventing proteasomal degradation, and modification of the nonconserved cysteine at the mIFITM1 C terminus supports an intramembrane topology with mechanistic implications.

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment.

BACKGROUND: Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS: We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPß. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPß. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 µM). CONCLUSION: The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

The IFITM proteins inhibit HIV-1 infection.

Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.