Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain

Voltage-gated calcium channels (Ca(V)s) govern muscle contraction, hormone and neurotransmitter release, neuronal migration, activation of calcium-dependent signalling cascades, and synaptic input integration. An essential Ca(V) intracellular protein, the beta-subunit (Ca(V)beta), binds a conserved domain (the alpha-interaction domain, AID) between transmembrane domains I and II of the pore-forming alpha(1) subunit and profoundly affects multiple channel properties such as voltage-dependent activation, inactivation rates, G-protein modulation, drug sensitivity and cell surface expression. Here, we report the high-resolution crystal structures of the Ca(V)beta2a conserved core, alone and in complex with the AID. Previous work suggested that a conserved region, the beta-interaction domain (BID), formed the AID-binding site; however, this region is largely buried in the Ca(V)beta core and is unavailable for protein-protein interactions. The structure of the AID-Ca(V)beta2a complex shows instead that Ca(V)beta2a engages the AID through an extensive, conserved hydrophobic cleft (named the alpha-binding pocket, ABP). The ABP-AID interaction positions one end of the Ca(V)beta near the intracellular end of a pore-lining segment, called IS6, that has a critical role in Ca(V) inactivation. Together, these data suggest that Ca(V)betas influence Ca(V) gating by direct modulation of IS6 movement within the channel pore.     

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.     

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin alpha and beta subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin alpha(IIb)beta3. Allostery in the beta3 I domain alters three metal binding sites, associated loops and alpha1- and alpha7-helices. Piston-like displacement of the alpha7-helix causes a 62 degrees reorientation between the beta3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper beta3 leg causes a 70 A separation between the knees of the alpha and beta legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.     

The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes.     

Structure of the apoptotic protease-activating factor 1 bound to ADP

Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.     

3BP2的SH2结构域的晶体结构和与来源于FRS2的肽的复合物的晶体结构

3BP2最初被作为一个Abl SH3结合蛋白被分离,但是其功能并不确定。除了富含脯氨酸区域和间接与SH3结合外,3BP2还有一个PH和Src同源区2结构域(SH2)。 Src同源区2结构域(SH2)是一个很大的家族,它们通过人体基因组编码的模块间的相互作用来识别酪氨酸磷酸化序列,由此在细胞信号转导和控制中发挥中心作用。肽基可以被SH2识别从而形成一种复合物.这篇文章的内容是关于3BP2的SH2结构域的晶体结构和与来源于FRS2的肽的复合物的晶体结构。依照表面电荷性质,这个结合袋的特异性是半极性半中性的。这个结构的特点明显的表现在,对于亲和性来说,Glu(p+1)比Ala (p+1) 或 Val (p+1)更为重要。     

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the alpha 3 domain of MHC I molecules.

The alpha 1 and alpha 2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the alpha 3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CD8. CD8 is important in the selection of T cells as anti-CD8 antibody injected into perinatal mice interferes with this process. We previously used a hybrid class I molecule with the alpha 1/alpha 2 domains from Ld and the alpha 3 domain from Q7b and showed that this molecule binds an Ld-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-Ld cytotoxic T lymphocytes. In addition, positive selection of virus-specific Ld-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the alpha 1/alpha 2 domains of class I, the alpha 3 domain plays an important part in both positive and negative selection of antigen-specific cells.     

Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins.

Many hormones, neurotransmitters and growth factors, on binding to G protein-coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits.     

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

Atomic structure of the actin:DNase I complex

The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined by X-ray analysis at an effective resolution of 2.8 A and 3A, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the beta- or beta- and gamma-phosphates, respectively. The motif of a five-stranded beta sheet consisting of a beta meander and a right handed beta alpha beta unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.     

Crystal structure of a Src-homology 3 (SH3) domain.

The Src-homologous SH3 domain is a small domain present in a large number of proteins that are involved in signal transduction, such as the Src protein tyrosine kinase, or in membrane-cytoskeleton interactions, but the function of SH3 is still unknown (reviewed in refs 1-3). Here we report the three-dimensional structure at 1.8 A resolution of the SH3 domain of the cytoskeletal protein spectrin expressed in Escherichia coli. The domain is a compact beta-barrel made of five antiparallel beta-strands. The amino acids that are conserved in the SH3 sequences are located close to each other on one side of the molecule. This surface is rich in aromatic and carboxylic amino acids, and is distal to the region of the molecule where the N and C termini reside and where SH3 inserts into the alpha-spectrin chain. We suggest that a protein ligand binds to this conserved surface of SH3.     

Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor

Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site.     

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.     

Structural and evolutionary analysis of HLA-D-region products

The major histocompatibility complex (MHC)--HLA in man and H-2 in mouse--encodes two classes of cell-surface antigens involved in the immune response. The amino acid sequences have been determined for a number of these molecules. Class I antigens, typified by the HLA-ABC antigens, are composed of a 43,000-molecular weight (MW) glycosylated transmembrane polypeptide with three external domains (alpha 1, alpha 2 and alpha 3), of which the one nearest the membrane (alpha 3) is associated with a 12,000-MW nonglycosylated polypeptide, beta 2-microglobulin. The HLA-D-region or class II antigens, DR, DC and SB, are composed of two glycosylated transmembrane polypeptides, of MWs 34,000 (alpha-chain) and 28,000 (beta-chain). Both chains have two external domains which presumably associate with each other, alpha 2, beta 2 being membrane proximal and alpha 1, beta 1 N-terminal and membrane distal. All four membrane-proximal domains (class I alpha 3, beta 2-microglobulin, class II alpha 2 and beta 2) have amino acid sequences that show significant similarities with immunoglobulin constant-region domains. This, together with the similarly placed internal disulphide bonds, suggests they might have an immunoglobulin-like structure (Fig. 1). We have now used computer graphics techniques to predict a detailed three-dimensional structure for the membrane-proximal domains of the class II antigens (alpha 2 and beta 2) based on the known coordinates of immunoglobulin constant domains (Fig. 2). The transmembrane regions of class II antigens have been modelled as two alpha-helices packed together. The proposed structure accounts for conservation of amino acids and leads to evolutionary predictions.     

Structures of complement component C3 provide insights into the function and evolution of immunity

The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the alpha2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the alpha-chain, including movement of a critical interaction site through a ring formed by the domains of the beta-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.     

Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U)

During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspF(U) protein into the host cytoplasm. EspF(U) controls actin by activating members of the Wiskott-Aldrich syndrome protein (WASP) family. Here we show that EspF(U) binds to the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain (for verprolin homology, central hydrophobic and acidic regions). This interaction potently activates WASP and neural (N)-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD-EspF(U) complex, EspF(U) forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspF(U) activates WASP by competing directly for the VCA binding site on the GBD. This mechanism is distinct from that used by the eukaryotic activators Cdc42 and SH2 domains, which globally destabilize the GBD fold to release the VCA. Such diversity of mechanism in WASP proteins is distinct from other multimodular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspF(U) and Cdc42/SH2, plus high-affinity EspF(U) binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.     

PX domain and CD domain play different roles in localization and vacuolation of Sorting Nexin 10

Sorting nexins （SNXs） are PX domain containing proteins and essential for intracellular protein sorting, trafficking and signal transduction. The PX domains of SNXs can bind to various phosphorelated phosphoinositides （PIs） and target the host proteins to endosomes. Recently, we have reported that overexpression of SNX10 in mammalian cells could induce giant vacuoles. In this study, we aimed to identify regions in SNX10 critical for the vacuolation activity. We found that both the PX domain and the CD1 region were essential for vacuolation. We provided evidence that the PX domain was able to specifically bind to Ptdlns（3）P and target SNX10 to endosomes. A mutation in the 131 region of the PX domain （V15A） disrupted the Ptdlns（3）P binding ability and the endosomal localization of SNX10. However, correct subcellular localization alone was not sufficient for SNX10 to induce vacuoles. We found that the CD1 region, which was not required for the localization, was indispensable for the vacuolation activity of SNX10. In summary, both the PX domain and the CD1 region are necessary for SNX10 to induce vacuoles but they play different roles in this process.     

Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A

The crystal structure of the RNA binding domain of the U1 small nuclear ribonucleoprotein A, which forms part of the ribonucleoprotein complex involved in the excision of introns, has been solved. It contains a four-stranded beta sheet and two alpha helices. The highly conserved segments designated RNP1 and RNP2 lie side by side on the middle two beta strands. U1 RNA binding studies of mutant proteins suggest that the RNA binds to the four-stranded beta sheet and to the flexible loops on one end.