Crystal clear: visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies

Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut)

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.

Crystal structures of the two membrane-proximal Ig-like domains (D3D4) of LILRB1/B2: alternative models for their involvement in peptide-HLA binding

Leukocyte immunoglobulin-like receptors (LILRs), also called CD85s, ILTs, or LIRs, are important mediators of immune activation and tolerance that contain tandem immunoglobulin (Ig)-like folds. There are 11 (in addition to two pseudogenes) LILRs in total, two with two Ig-like domains (D1D2) and the remaining nine with four Ig-like domains (D1D2D3D4). Thus far, the structural features of the D1D2 domains of LILR proteins are well defined, but no structures for the D3D4 domains have been reported. This is a very important field to be studied as it relates to the unknown functions of the D3D4 domains, as well as their relative orientation to the D1D2 domains on the cell surface. Here, we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2. The two Ig-like domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle (∼60°) to form a bent structure, resembling the structures of natural killer inhibitory receptors. Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures, two alternative models of full-length (four Ig-like domains) LILR molecules bound to HLA I are proposed.

An octamer of enolase from Streptococcus suis

Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells. The enzyme can also locate on the cell surface and bind to plasminogen, via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells. The functions of the eukaryotic enzymes on the cell surface expression (including T cells, B cells, neutrophils, monocytoes, neuronal cells and epithelial cells) are not known. Streptococcus suis serotype 2 (S. suis 2, SS2) is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome (STSS) never seen before in human sufferers. We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection. In this study, a 2.4-angstrom structure of the SS2 enolase is solved, revealing an octameric arrangement in the crystal. We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay. These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well. This is, to our knowledge, the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically, and the second octamer enolase structure in addition to that of Streptococcus pneumoniae. We also investigated the plasminogen binding property of the SS2 enzyme.

X-ray diffraction analysis of glycoprotein D from herpes simplex virus type 2 / 生物工程学报

Glycoprotein D (gD) of Herpes simplex virus type 2 (HSV-2) is a key factor mediating the entry of HSV-2 into host cells. In order to explain the mechanism underlying the gD-mediated receptor-binding and viral entry, we performed a structural study on HSV-2 gD. The ectodomain of the gD protein encompassing residues 1 to 285 was expressed by baculovirus-infected insect cells as a secreted soluble protein with a C-terminal hexa-his tag. The protein was then purified by affinity and size-exclusion chromatography. The purified protein was successfully crystallized using the hanging-drop vapor-diffusion at 18 degrees C in a condition consisting of 0.1 mol/L Hepes pH 7.2, 5% (V/V) 2-methyl-2,4-pentanediol (MPD) and 10% PEG 10 000. The crystals diffracted to 1.8 angstroms resolution and belonged to space group P21, with unit-cell parameters alpha = 63.6, b = 55.4, c = 65.3 angstroms, beta = 96.3 degrees.

Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines

There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs. The main component of the current split influenza A virus vaccine is viral hemagglutinin (HA) which induces a strong antibody-mediated immune response. To develop a modern vaccine against influenza A viruses, the current research has been focused on the universal vaccines targeting viral M2, NP and HA proteins. Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface, and each monomer consists of a globular head (HA1) and a "rod-like" stalk region (HA2), the latter being more conserved among different HA subtypes and being the primary target for universal vaccines. In this study, we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A (H1N1) virus HA as a model, tested its immunogenicity in mice, solved its crystal structure and further examined its immunological characteristics. The results show that the HA globular head can be easily prepared by in vitro refolding in an E. coli expression system, which maintains its intact structure and allows for the stimulation of a strong immune response. Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.

Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus

Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence analysis of the 2009 S-OIV hemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, analysis at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of soluble hemagglutinin H1 (09H1) at 2.9 Å, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby (Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.

A dimeric structure of PD-L1: functional units or evolutionary relics?

PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222(1) with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.

Expression and crystallographic studies of a fungal immunomodulatory protein LZ-8 from a medicinal fungus Ganoderma lucidum / 生物工程学报

LZ-8 protein, isolated from a well known Chinese traditional medicinal fungus Ganoderma lucidum, is the first member of fungal immunomodulatory protein, members of which have been isolated from a variety of medicinal and edible mushrooms in the last two decades. The protein plays a multifunctional and important role in modulating immune system. In this report, in order to get LZ-8 protein crystals, the LZ-8 gene was expressed and purified by affinity chromatography, gel filtration chromatography and anion exchange chromatography subsequently. The protein was then crystallized using the hanging-drop vapour-diffusion method. The LZ-8 crystals were obtained and the phase information was calculated by X-ray diffraction. The resolution of LZ-8 crystals is 3.2A. This study will provide an insight into the structure of fungal immunomodulatory proteins.

Purification, crystallographic analysis of rhesus MHC-I Mamu-A*02 complexed with simian immunodeficiency virus nonapeptide / 生物工程学报

Rhesus macaque (Macaca mulatta) is the best model to study of human immunodeficiency virus (HIV) infection and to develop acquired immunodeficiency syndrome (AIDS) vaccine. The crystal structure of its major histocompatibility antigen complex (MHC) is helpful to understand the mechanism of HIV immune evasion. In this study, we cloned the light chain (beta2m) of MHC class I allele of rhesus macaques, Mamu-A*02, and inserted it into pET21a(+) vector. We transfected the recombinant plasmid pET21a(+)-Mamu-beta2m and pET21a(+)-Mamu-alpha into BL21(DE3). Mamu-A*02 and beta2m were expressed in the form of inclusion bodies in BL21 (DE3). We co-refolded the inclusion bodies of Mamu-alpha and Mamu-beta2m with SIV nonapeptide YY9 and obtained the correct refolded protein complex. Then we purified the protein complex by the gel filtration and anion-exchange column. With hanging-drop method, we screened and optimized for the protein crystal. We managed to collect a X-ray diffraction with the resolution to 2.8 angstroms in the condition of 0.1 mol/L BIS-TRIS (pH5.5), 2.0 mol/L(NH4)2SO4. This crystal belong to perpendicular space group P2(1)2(1)2(1), with unit-cell parameters a = 128.99 angstroms, b = 129.01 angstroms, c = 129.03 angstroms. This data is available for the structure determination.