Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2D(d) through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2D(d). The Ly49A dimer interacts extensively with two H-2D(d) molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2D(d) alpha2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.     

Segregation of receptor and ligand regulates activation of epithelial growth factor receptor

Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its receptors--erbB2, erbB3 and erbB4 (refs 1-3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand-receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-alpha is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2-4, which segregate to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-alpha activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes ('polarized'), or when tight junctions between adjacent cells are opened, heregulin-alpha activates its receptor. This mechanism of ligand-receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.     

Argos inhibits epidermal growth factor receptor signalling by ligand sequestration

The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.     

Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor.

Nerve growth factor (NGF) is involved in a variety of processes involving signalling, such as cell differentiation and survival, growth cessation and apoptosis of neurons. These events are mediated by NGF as a result of binding to its two cell-surface receptors, TrkA and p75. TrkA is a receptor with tyrosine kinase activity that forms a high-affinity binding site for NGF. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding. Here we present the crystal structure of human NGF in complex with human TrkA-d5 at 2.2 A resolution. The ligand-receptor interface consists of two patches of similar size. One patch involves the central beta-sheet that forms the core of the homodimeric NGF molecule and the loops at the carboxy-terminal pole of TrkA-d5. The second patch comprises the amino-terminal residues of NGF, which adopt a helical conformation upon complex formation, packing against the 'ABED' sheet of TrkA-d5. The structure is consistent with results from mutagenesis experiments for all neurotrophins, and indicates that the first patch may constitute a conserved binding motif for all family members, whereas the second patch is specific for the interaction between NGF and TrkA.     

Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region

A domain in the low-density lipoprotein receptor contains three cysteine-rich 'growth factor' repeats like those that occur in many proteins. When this domain is deleted, the receptor no longer releases its ligand at acid pH, it is no longer recycled efficiently and it is rapidly degraded after ligand binding.     

Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate

Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC-FimH chaperone-adhesin complex and that of the unbound form of the FimD translocation domain. The FimD-FimC-FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC-FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the β-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone-subunit complex. The FimD-FimC-FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.     

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 alpha1 and alpha2 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.     

Basic fibroblast growth factor fused to a signal peptide transforms cells

Basic fibroblast growth factor (bFGF) is a potent growth and angiogenic factor that is found in abundance in tissues such as brain, hypothalamus, kidney and cartilage. Despite this copious production of bFGF, most of these tissues are not undergoing either active growth or angiogenesis, suggesting that bFGF activity must be regulated so as to prevent autostimulation of cell growth. In cultured cells, bFGF is associated mainly with cells and basement membranes and is not released into the medium. Prevention of release could be a mechanism for regulation of bFGF activity and may be a consequence of the apparent absence of a secretory-signal sequence in the bFGF protein. Here we investigate whether this regulation can be overridden through the forced secretion of bFGF. Such secretion might provide the bFGF access to its receptor and in turn lead to autocrine transformation of the cell. We report that bFGF, as specified by a recombinant plasmid, is itself unable to induce such transformation, but acquires this ability after fusion with a secretory-signal sequence. The resulting transformants undergo unusual morphological alteration and display tumorigenicity.     

Crystal structure of a streptococcal protein G domain bound to an Fab fragment.

Protein G is a cell-surface protein from Streptococcus which binds to IgG molecules from a wide range of species with an affinity comparable to that of antigen. The high affinity of protein G for the Fab portion of IgG poses a particular challenge in molecular recognition, given the variability of heavy chain subclass, light chain type and complementarity-determining regions. Here we report the crystal structure of a complex between a protein G domain and an immunoglobulin Fab fragment. An outer beta-strand in the protein G domain forms an antiparallel interaction with the last beta-strand in the constant heavy chain domain of the immunoglobulin, thus extending the beta-sheet into the protein G. The interaction between secondary structural elements in Fab and protein G provides an ingenious solution to the problem of maintaining a high affinity for many different IgG molecules. The structure also contrasts with Fab-antigen complexes, in which all contacts with antigen are mediated by the variable regions of the antibody, and to our knowledge provides the first details of interaction of the constant regions of Fab with another protein.     

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.     

Structure of hisactophilin is similar to interleukin-1 beta and fibroblast growth factor.

The fast reaction of the actin-based cytoskeleton in motile cells after stimulation with a chemoattractant requires a signal-transduction chain that creates a very specific environment at distinct regions beneath the plasma membrane. Dictyostelium hisactophilin, a unique actin-binding protein, is a submembranous pH sensor that signals slight changes of the H+ concentration to actin by inducing actin polymerization and binding to microfilaments only at pH values below seven. It has a relative molecular mass of 13.5K and its most unusual feature is the presence of 31 histidine residues among its total of 118 amino acids. The transduction of an external signal from the plasma membrane to the cytoskeleton is poorly understood. Here we report the protein's structure in solution determined by nuclear magnetic resonance spectroscopy. The nuclear Overhauser effect intensities of the three-dimensional nuclear Overhauser spectra were used directly in the calculations. The overall folding of histactophilin is similar to that of interleukin-1 beta and fibroblast growth factor, but the primary amino-acid sequence of hisactophilin is unrelated to these two proteins.     

Crystal structure of the calcium pump with a bound ATP analogue

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across cell and organelle membranes. Here, we describe the crystal structure of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum, a representative member of the P-type ATPase superfamily, with an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites. In this state, the ATP analogue reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the transmembrane helices moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum.     

重组人碱性成纤维细胞生长因子的纯化及生物活性鉴定

目的 :探讨重组人碱性成纤维细胞生长因子 (rhbFGF)的纯化工艺和活性鉴定 .方法 :将rhbFGF工程菌大量扩增后通过包涵体提取、复性、阳离子交换、凝胶过滤等技术进行纯化 .结果 :纯化后的rhbFGF ,相对分子质量为 17× 10 3,蛋白纯度为 95 %以上 ,比活为 1 7× 10 6 U/mg ,对NIH3T3细胞具有明显的促分裂活性 .结论 :通过复性和两步层析的纯化方法可获得高纯度、高回收率和高生物活性的rhbFGF ,可用于实验室研究及临床试验     

Crystal structure of the ligand-free G-protein-coupled receptor opsin

In the G-protein-coupled receptor (GPCR) rhodopsin, the inactivating ligand 11-cis-retinal is bound in the seven-transmembrane helix (TM) bundle and is cis/trans isomerized by light to form active metarhodopsin II. With metarhodopsin II decay, all-trans-retinal is released, and opsin is reloaded with new 11-cis-retinal. Here we present the crystal structure of ligand-free native opsin from bovine retinal rod cells at 2.9 ?ngstr?m (A) resolution. Compared to rhodopsin, opsin shows prominent structural changes in the conserved E(D)RY and NPxxY(x)(5,6)F regions and in TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 A, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, some of which were attributed to an active GPCR state, reorganize the empty retinal-binding pocket to disclose two openings that may serve the entry and exit of retinal. The opsin structure sheds new light on ligand binding to GPCRs and on GPCR activation.     

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.