Peter D. Sun

Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand.

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the α1 and α2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor–ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling

Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-β/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3-deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3(-)(/-) white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3(-/-) adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-1α expression. We observe significant correlation between TGF-β1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-β signaling protects mice from obesity, diabetes, and hepatic steatosis. Together, these results demonstrate that TGF-β signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-β activity might be an effective treatment strategy for obesity and diabetes.

Structural recognition and functional activation of FcγR by innate pentraxins

Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway through C1q (refs 3 and 4). More recently, members of the pentraxin family were found to interact with cell-surface Fcγ receptors (FcγR) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin’s binding to FcγR and its functional activation of FcγR-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and FcγRIIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and FcγRIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for FcγR isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for FcγR binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the FcγR pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.

A STRUCTURAL PERSPECTIVE ON MHC CLASS I RECOGNITION BY KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTORS

Killer cell immunoglobulin-like receptors (KIR) play a critical role in the regulation of natural killer (NK) cell activity through their recognition of class I MHC molecules expressed on target cells. KIR recognition provides vital information to NK cells about whether a target cell should be lysed or spared. Understanding the molecular mechanism of this recognition has remained a strong focus of investigation. This has resulted in the crystal structures of several members of the KIR family and more recently the determinations of the three dimensional structures of KIR2DL2 and KIR2DL1 complexed with their respective ligands, HLA-Cw3 and HLA-Cw4. A strong structural conservation has been revealed both in the receptor design and in the overall mode of KIR binding to class I molecules. Nevertheless, distinct differences in the receptor binding sites allow for high specificity between ligands. Furthermore, unexpected similarities with T-cell receptor (TCR) recognition of MHC molecules are also observed. The detailed interactions between KIR and HLA-C molecules and their functional implications will be reviewed here.

CONFORMATIONAL PLASTICITY REVEALED BY THE COCRYSTAL STRUCTURE OF NKG2D AND ITS CLASS I MHC-LIKE LIGAND ULBP3

NKG2D is known to trigger the natural killer (NK) cell lysis of various tumor and virally infected cells. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles the alpha1 and alpha2 domains of classical MHC molecules without a bound peptide. The lack of alpha3 and beta2m domains is compensated by replacing two hydrophobic patches at the underside of the class I MHC-like beta sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 alpha helices, creating a complementary interface, an asymmetrical subunit orientation, and local conformational adjustments in the receptor. The interface is stabilized primarily by hydrogen bonds and hydrophobic interactions. Unlike the KIR receptors that recognize a conserved HLA region by a lock-and-key mechanism, NKG2D recognizes diverse ligands by an induced-fit mechanism.

Structure of CD94 Reveals a Novel C-Type Lectin Fold: Implications for the NK Cell–Associated CD94/NKG2 Receptors

The crystal structure of the extracellular domain of CD94, a component of the CD94/NKG2 NK cell receptor, has been determined to 2.6 A resolution, revealing a unique variation of the C-type lectin fold. In this variation, the second alpha helix, corresponding to residues 102-112, is replaced by a loop, the putative carbohydrate-binding site is significantly altered, and the Ca2+-binding site appears nonfunctional. This structure may serve as a prototype for other NK cell receptors such as Ly-49, NKR-P1, and CD69. The CD94 dimer observed in the crystal has an extensive hydrophobic interface that stabilizes the loop conformation of residues 102-112. The formation of this dimer reveals a putative ligand-binding region for HLA-E and suggests how NKG2 interacts with CD94.

Structure of killer cell immunoglobulin-like receptors and their recognition of the class I MHC molecules

Summary: The recognition of class I MHC molecules by killer cell immunoglobulin‐like receptors (KIR) constitutes an integral part of immune surveillance by the innate immune system. To understand the molecular basis of this recognition, the structures of several members of this superfamily have been determined. Despite their functional diversity, members of this superfamily share many conserved structural features. A central question is how these receptors recognize their ligands. The recent determination of the crystal structure of KIR2DL2 in complex with HLA‐Cw3 has revealed the molecular mechanisms underpinning this interaction, which ultimately modulates the cytolytic activity of natural killer cells. While the recognition of MHC molecules by KIR is characterized by a number of unique features, some unexpected similarities with T‐cell receptor recognition of MHC molecules are also observed. The detailed interactions between KIR2DL2 and HLA‐Cw3 and their functional implications will be reviewed here.

Crystal Structure of the Extracellular Domain of a Human FcγRIII

Fc receptors play a major role in immune defenses against pathogens and in inflammatory processes. The crystal structure of a human immunoglobulin receptor, FcgammaRIIIb, has been determined to 1.8 A resolution. The overall fold consists of two immunoglobulin-like domains with an acute interdomain hinge angle of approximately 50 degrees. Trp-113, wedged between the N-terminal D1 and the C-terminal D2 domains, appears to further restrict the hinge angle. The putative Fc binding region of the receptor carries a net positive charge complementary to the negative-charged receptor binding regions on Fc. A 1:1 binding stoichiometry between the receptor and Fc was measured by both the equilibrium and nonequilibrium size-exclusion chromatography. Two separate parallel dimers are observed in the crystal lattice, offering intriguing models for receptor aggregation.

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.

Characterization of DC-SIGN/R Interaction with Human Immunodeficiency Virus Type 1 gp120 and ICAM Molecules Favors the Receptor's Role as an Antigen-Capturing Rather than an Adhesion Receptor

ABSTRACT The dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin binding receptor (DC-SIGN) was shown to bind human immunodeficiency virus type 1 (HIV-1) viral envelope protein gp120 and proposed to function as a Trojan horse to enhance trans-virus infection to host T cells. To better understand the mechanism by which DC-SIGN and DC-SIGNR selectively bind HIV-1 gp120, we constructed a series of deletion mutations in the repeat regions of both receptors. Different truncated receptors exist in different oligomeric forms. The carbohydrate binding domain without any repeats was monomeric, whereas the full extracellular receptors existed as tetramers. All reconstituted receptors retained their ability to bind gp120. The dissociation constant, however, differed drastically from micromolar values for the monomeric receptors to nanomolar values for the tetrameric receptors, suggesting that the repeat region of these receptors contributes to the avidity of gp120 binding. Such oligomerization may provide a mechanism for the receptor to selectively recognize pathogens containing multiple high-mannose-concentration carbohydrates. In contrast, the receptors bound to ICAMs with submicromolar affinities that are similar to those of two nonspecific cell surface glycoproteins, FcγRIIb and FcγRIII, and the oligomerization of DC-SIGNR resulted in no increase in binding affinity to ICAM-3. These findings suggest that DC-SIGN may not discriminate other cell surface glycoproteins from ICAM-3 binding. The pH dependence in DC-SIGN binding to gp120 showed that the receptor retained high-affinity gp120 binding at neutral pH but lost gp120 binding at pH 5, suggesting a release mechanism of HIV in the acidic endosomal compartment by DC-SIGN. Our work contradicts the function of DC-SIGN as a Trojan horse to facilitate HIV-1 infection; rather, it supports the function of DC-SIGN/R (a designation referring to both DC-SIGN and DC-SIGNR) as an antigen-capturing receptor.