Structure of a C-type mannose-binding protein complexed with an oligosaccharide.

C-type (Ca(2+)-dependent) animal lectins such as mannose-binding proteins mediate many cell-surface carbohydrate-recognition events. The crystal structure at 1.7 A resolution of the carbohydrate-recognition domain of rat mannose-binding protein complexed with an oligomannose asparaginyl-oligosaccharide reveals that Ca2+ forms coordination bonds with the carbohydrate ligand. Carbohydrate specificity is determined by a network of coordination and hydrogen bonds that stabilizes the ternary complex of protein, Ca2+ and sugar. Two branches of the oligosaccharide crosslink neighbouring carbohydrate-recognition domains in the crystal, enabling multivalent binding to a single oligosaccharide chain to be visualized directly.     

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.     

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.     

Optimization of specificity in a cellular protein interaction network by negative selection

Most proteins that participate in cellular signalling networks contain modular protein-interaction domains. Multiple versions of such domains are present within a given organism: the yeast proteome, for example, contains 27 different Src homology 3 (SH3) domains. This raises the potential problem of cross-reaction. It is generally thought that isolated domain-ligand pairs lack sufficient information to encode biologically unique interactions, and that specificity is instead encoded by the context in which the interaction pairs are presented. Here we show that an isolated peptide ligand from the yeast protein Pbs2 recognizes its biological partner, the SH3 domain from Sho1, with near-absolute specificity--no other SH3 domain present in the yeast genome cross-reacts with the Pbs2 peptide, in vivo or in vitro. Such high specificity, however, is not observed in a set of non-yeast SH3 domains, and Pbs2 motif variants that cross-react with other SH3 domains confer a fitness defect, indicating that the Pbs2 motif might have been optimized to minimize interaction with competing domains specifically found in yeast. System-wide negative selection is a subtle but powerful evolutionary mechanism to optimize specificity within an interaction network composed of overlapping recognition elements.     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

Structure and evolution of ricin B chain

Ricin is a dimeric toxin from the castor bean Ricinus communis, which is composed of a sugar-binding subunit (B) that attaches to receptors on the surfaces of target cells and a subunit (A) with enzymatic activity that attacks and inactivates ribosomes. We report here that comparison of amino-acid sequence data with high-resolution structure analysis of the ricin B subunit shows it to be the product of a series of gene duplications. The modern protein has two sugar-binding domains, each of which is composed of three copies of a more ancient galactose-binding peptide of about 40 residues.     

DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA

Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.     

Bioinformatics analysis of two-component regulatory systems in Staphylococcus epidermidis

Sixteen pairs of two-component regulatory systems are identified in the genome of Staphylococcus epidermidis ATCC12228 strain, which is newly sequenced by our laboratory for Medical Molecular Virology and Chinese National Human Genome Center at Shanghai, by using bioinformatics analysis. Comparative analysis of the twocomponent regulatory systems in S. epidermidis and that of S.aureus and Bacillus sublilis shows that these systems may regulate some important biological functions, e.g. growth,biofilm formation, and expression of virulence factors in S.epidermidis. Two conserved domains, i.e. HATPase_c and REC domains, are found in all 16 pairs of two-component proteins.Homologous modelling analysis indicates that there are 4 similar HATPase_c domain structures of histidine kinases and 13 similar REC domain structures of response regulators,and there is one AMP-PNP binding pocket in the HATPase_c domain and three active aspartate residues in the REC domain. Preliminary experiment reveals that the bioinformatics analysis of the conserved domain structures in the two-component regulatory systems in S. epidermidis may provide useful information for discovery of potential drug target.     

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

The proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.     

石蒜凝集素基因的克隆

用石蒜科植物凝集素基因的保守序列为引物，利用RACE－PCR技术从石蒜幼嫩叶片中克隆出石蒜凝集素的全长cDNA，通过比较石蒜同其他石蒜科植物凝集素基因序列和推测的氨基酸序列，发现石蒜凝集素基因编码一具有信号肽的前体蛋白，石蒜凝集素同其他石蒜科植物凝集素一样为具有3个甘露糖专一结合盒的凝集素。     

The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4

CD4, a cell-surface glycoprotein expressed on a subset of T-cells and macrophages, serves as the receptor for the human immunodeficiency virus (HIV) (reviewed in ref. 1), binding to the HIV envelope glycoprotein, gp120 with high affinity. Attempts to block infection in vivo by raising antibodies against gp120 have failed, probably because these antibodies have insufficient neutralizing activity. In addition, because of the extensive polymorphism of gp120 in different isolates of HIV, antibodies raised against one HIV isolate are only weakly effective against others. Because interaction with CD4 is essential for infectivity by all isolates of HIV, an agent that could mimic CD4 in its ability to bind to gp120, such as a peptide or monoclonal antibody, might block infection by a wide spectrum of isolates. To aid the identification of such a ligand we have defined regions of CD4 that are required for binding to gp120. Although human CD4 is similar to mouse CD4 in amino-acid sequence (55% identity, ref. 6) and structure, we have found that the murine protein fails to bind detectably to gp120 and have exploited this finding to study binding of gp120 to mouse-human chimaeric CD4 molecules. These studies show that amino-acid residues within the amino-terminal immunoglobulin-like domain of human CD4 are involved in binding to gp120 as well as to many anti-CD4 monoclonal antibodies.     

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.     

Ligand-receptor binding revealed by the TNF family member TALL-1

The tumour necrosis factor (TNF) ligand TALL-1 and its cognate receptors, BCMA, TACI and BAFF-R, were recently identified as members of the TNF superfamily, which are essential factors contributing to B-cell maturation. The functional, soluble fragment of TALL-1 (sTALL-1) forms a virus-like assembly for its proper function. Here we determine the crystal structures of sTALL-1 complexed with the extracellular domains of BCMA and BAFF-R at 2.6 and 2.5 A, respectively. The single cysteine-rich domain of BCMA and BAFF-R both have saddle-like architectures, which sit on the horseback-like surface formed by four coil regions on each individual sTALL-1 monomer. Three novel structural modules, D2, X2 and N, were revealed from the current structures. Sequence alignments, structural modelling and mutagenesis revealed that one disulphide bridge in BAFF-R is critical for determining the binding specificity of the extracellular domain eBAFF-R to TALL-1 instead of APRIL, a closely related ligand of TALL-1, which was confirmed by binding experiments in vitro.     

Structural basis for EGFR ligand sequestration by Argos

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-beta family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.