Zhongcheng Zou

Ternary Complex of Transforming Growth Factor-β1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily

Transforming growth factor (TGF)-β1, -β2, and -β3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-Å resolution crystal structure of the ternary complex between human TGF-β1 and the extracellular domains of its type I and type II receptors, TβRI and TβRII. The TGF-β1 ternary complex structure is similar to previously reported TGF-β3 complex except with a 10° rotation in TβRI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-β. TβRI showed significant binding to TGF-β2 and TGF-β3 but not TGF-β1, and the binding to all three isoforms of TGF-β was enhanced considerably in the presence of TβRII. The preference of TGF-β2 to TβRI suggests a variation in its receptor recruitment in vivo. Although TGF-β1 and TGF-β3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-β superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of TβRII on TGF-β is unique to TGF-βs, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

Natural killer cell-produced IFN-γ and TNF-α induce target cell cytolysis through up-regulation of ICAM-1

NK cells control tumor and virus‐infected cells through releasing cytotoxic granules and proinflammatory cytokines. IFN‐γ and TNF‐α secretions and cytotoxicity are regarded as two distinct functions of NK cells with little synergy in between as results of early association of the two functions with distinct subsets of NK populations and of the studies showing target cells developing NK resistance upon IFN‐γ treatment. Here, we show that IFN‐γ and TNF‐α synergistically enhance NK cell cytotoxicity through NF‐κB‐dependent up‐regulation of ICAM‐1 expression in target cells, thereby promoting their conjugate formation with NK cells. Neutralizing IFN‐γ and TNF‐α during cytolysis significantly impaired NK cell lysis of the target cells. Further, tumor cells exhibiting IFN‐γ‐inducible lysis are generally less‐sensitive NK target cells but express inducible levels of ICAM‐1. In contrast, sensitive NK targets tend to express higher but less‐inducible ICAM‐1. Their preferential induction in the lysis of insensitive NK target cells suggests that IFN‐γ and TNF‐α are functionally linked to and should be regarded as an integral part of NK cytolytic function.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

Significance Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. It is the sole high-affinity receptor for IgG and plays a significant role in immunotherapy. To date, there is no structural information available on how the receptor recognizes its antibody ligands, however. Consequently, the mechanism of its high-affinity IgG binding remains unclear. We report the first structure of the high-affinity Fc receptor in complex with IgG-Fc. The structural work reveals a direct receptor recognition of Fc glycan as a major factor in receptor affinity. This is the first example of Fc receptor making direct glycan contact through protein residues. The results have implications for the use of glycan engineering in immunotherapy. Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop–glycan interaction resulted in an ∼20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

Siglecs facilitate HIV-1 infection of macrophages through adhesion with viral sialic acids.

Background Human immunodeficiency virus type 1 (HIV-1) infects macrophages effectively, despite relatively low levels of cell surface-expressed CD4. Although HIV-1 infections are defined by viral tropisms according to chemokine receptor usage (R5 and X4), variations in infection are common within both R5- and X4-tropic viruses, indicating additional factors may contribute to viral tropism. Methodology and Principal Findings Using both solution and cell surface binding experiments, we showed that R5- and X4-tropic HIV-1 gp120 proteins recognized a family of I-type lectin receptors, the Sialic acid-binding immunoglobulin-like lectins (Siglec). The recognition was through envelope-associated sialic acids that promoted viral adhesion to macrophages. The sialic acid-mediated viral-host interaction facilitated both R5-tropic pseudovirus and HIV-1BaL infection of macrophages. The high affinity Siglec-1 contributed the most to HIV-1 infection and the variation in Siglec-1 expression on primary macrophages from different donors was associated statistically with sialic acid-facilitated viral infection. Furthermore, envelope-associated sialoglycan variations on various strains of R5-tropic viruses also affected infection. Conclusions and Significance of the Findings Our study showed that sialic acids on the viral envelope facilitated HIV-1 infection of macrophages through interacting with Siglec receptors, and the expression of Siglec-1 correlated with viral sialic acid-mediated host attachment. This glycan-mediated viral adhesion underscores the importance of viral sialic acids in HIV infection and pathogenesis, and suggests a novel class of antiviral compounds targeting Siglec receptors.

Making Sense of the Diverse Ligand Recognition by NKG2D

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.

Structural and Functional Studies of Igαβ and Its Assembly with the B Cell Antigen Receptor

The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Igbeta form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Igalpha displays a similar fold as Igbeta. An Igalphabeta heterodimer model was generated based on the unique disulfide-bonded Igbeta dimer. Solution binding studies showed that the extracellular domains of Igalphabeta preferentially recognize the constant region of BCR with mu chain specificity, suggesting a role for Igalphabeta to enhance BCRmu chain signaling. Cluster mutations on Igalpha, Igbeta, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the Cmu4 domain of mIgM. These studies provide the first structural model for understanding BCR function.

Influence of the second amino acid on recombinant protein expression.

Factors affecting protein expression have been intensely studied to the benefit of recombinant protein production. Through mutational analysis at the +2 amino acid position of recombinant Igα, we examined the effect of all 20 amino acids on protein expression. The results showed that amino acids at the +2 position affected 10-fold in the recombinant protein expression. Specifically, Ala, Cys, Pro, Ser, Thr, and Lys at the +2 position resulted in significantly higher expression of recombinant Igα than other amino acids, while Met, His and Glu resulted in greatly reduced protein expression. This expression difference depended on the amino acid instead of their codon usage. Consistent with the mutational results, a statistically significant enrichment in Ala and Ser at the +2 position was observed among highly expressed Escherichia coli genes. This work suggests a general approach to enhance protein expression by incorporating an Ala or Ser after the initiation codon.

Development of an improved mammalian overexpression method for human CD62L.

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5-30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.

HIV-1 targets L-selectin for adhesion and induces its shedding for viral release

CD4 and chemokine receptors mediate HIV-1 attachment and entry. They are, however, insufficient to explain the preferential viral infection of central memory T cells. Here, we identify L-selectin (CD62L) as a viral adhesion receptor on CD4+ T cells. The binding of viral envelope glycans to L-selectin facilitates HIV entry and infection, and L-selectin expression on central memory CD4+ T cells supports their preferential infection by HIV. Upon infection, the virus downregulates L-selectin expression through shedding, resulting in an apparent loss of central memory CD4+ T cells. Infected effector memory CD4+ T cells, however, remain competent in cytokine production. Surprisingly, inhibition of L-selectin shedding markedly reduces HIV-1 infection and suppresses viral release, suggesting that L-selectin shedding is required for HIV-1 release. These findings highlight a critical role for cell surface sheddase in HIV-1 pathogenesis and reveal new antiretroviral strategies based on small molecular inhibitors targeted at metalloproteinases for viral release.HIV binding is mediated via CD4 and chemokine co-receptors, but this does not explain the preferential infection of central memory CD4+ T cells. Here the authors show HIV targets L-selectin, induces shedding from the infected cell, and inhibition of L-selectin reduces HIV infection and release.