Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes

Factor VIII is generally believed to circulate in blood as a multimeric complex of two glycoproteins which are physiologically and immunologically distinct. One component of the factor VIII complex is factor VIII procoagulant activity (FVIII:C) which is associated with factor VIII/procoagulant antigen (FVIII:Ag, formerly FVIII/CAg). The second, larger unit of the complex is factor VIII/von Willebrand factor (vWF:Ag, formerly factor VIII-related antigen or FVIIIRAg). FVIII:C has anti-haemophilic activity and is defective or deficient in patients with classical haemophilia, and vWF:Ag is absent in patients with von Willebrand disease. FVIII:Ag was demonstrated recently in endothelial cells lining hepatic sinusoids, by using immunoperoxidase staining and light microscopy, whereas biochemical data had indicated its presence predominantly in the hepatocyte fractions and in lesser amounts in endothelial cells. Moreover, recent hybridization experiments detected FVIII:C messenger RNA in liver and kidney tissues. Despite several efforts, the cells responsible for FVIII:C synthesis have not been unequivocally identified. Here we use protein A-gold complex labelling to demonstrate the ultrastructural localization of FVIII:C in human liver cells; the results indicate that hepatocytes may synthesize FVIII:Ag.     

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.     

Structure of the pancreatic lipase-procolipase complex.

Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.     

Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain

RNA interference is a conserved mechanism that regulates gene expression in response to the presence of double-stranded (ds)RNAs. The RNase III-like enzyme Dicer first cleaves dsRNA into 21-23-nucleotide small interfering RNAs (siRNAs). In the effector step, the multimeric RNA-induced silencing complex (RISC) identifies messenger RNAs homologous to the siRNAs and promotes their degradation. The Argonaute 2 protein (Ago2) is a critical component of RISC. Both Argonaute and Dicer family proteins contain a common PAZ domain whose function is unknown. Here we present the three-dimensional nuclear magnetic resonance structure of the Drosophila melanogaster Ago2 PAZ domain. This domain adopts a nucleic-acid-binding fold that is stabilized by conserved hydrophobic residues. The nucleic-acid-binding patch is located in a cleft between the surface of a central beta-barrel and a conserved module comprising strands beta3, beta4 and helix alpha3. Because critical structural residues and the binding surface are conserved, we suggest that PAZ domains in all members of the Argonaute and Dicer families adopt a similar fold with nucleic-acid binding function, and that this plays an important part in gene silencing.     

SH2域与磷酸化多肽相互作用的连续溶剂模型研究

使用连续溶剂模型方法研究了SH2结构域与磷酸化多肽pYXXX(X为20种常见氨基酸残基中的任意一种)之间的相互作用.首先计算了已知的SH2域-磷酸化酪氨酸多肽复合物之间的结合能,理论计算的结果与实验测得的亲合能之间的相关系数为0.91,验证了理论模型的正确性.然后,用该模型方法计算了SH2域与pYXXX之间的结合能,分析了磷酸化酪氨酸多肽pYXXX中 1, 2, 3位置上残基对结合能的影响.结果表明 2, 3位置上残基的变化对结合能影响较大, 2位置上带负电的残基和 3位置上的疏水性残基有利于SH2域与pYXXX之间的相互作用,这与实验结果一致.     

Unfolding of C2A Domain of Synaptotagmin I in the Presence of Guanidine Hydrochloride

The C2 domain originally referred to the second of four constant structural motifs in protein kinase C (PKC). Now this domain represents a large structural family sharing a homologous dimensional structure in many proteins that play important roles in many organisms. The C2A domain is one of the two C2 domains of synaptotagmin I involved in the Ca^2 regulation of exocytosis. This domain is mostly composed of β-sheet except for a small fraction of α-helix, and therefore provides an ideal model for a protein folding study. In this report, the unfolding equilibrium of the C2A domain in guanidine hydrochloride (GdnHCI) containing solutions has been studied using ultraviolet (UV) difference spectrum, fluorescence spectrum, size exclusion chromatography (SEC), and circular dichroism (CD) spectrum. The results suggest that unfolding of the C2A domain occurs as a two-state process during GdnHCI titration. By examining the changes of both tertiary structure and secondary structure, no intermediates could be detected during this unfolding study. However, it has been found that the native state of the C2A domain has a large hydrophobic surface. This result suggests that as a fragment of a protein, the C2A domain itself may exist in a state with large hydrophobic surface. This hydrophobic surface may be the molecular basis for interaction between domains in the whole protein.Furthermore, the hydrophobic behavior may play a role during the oligomerization of svnaptotagmin.     

Enhancing protein C interaction with thrombin results in a clot-activated anticoagulant.

Human protein C is a vitamin K-dependent plasma glycoprotein that circulates as an inactive zymogen. At the endothelial cell surface, thrombin in complex with the integral membrane protein thrombomodulin converts protein C to its active form by specific cleavage of an activation peptide. The activated form of protein C has potent anticoagulant activity as a feedback regulator of thrombin generation (reviewed in refs 4-6), and also has profibrinolytic, anti-ischaemic and anti-inflammatory properties. Protein C is effective in the treatment of model and human thrombotic diseases but, except when it has been used to treat genetic or acquired deficiencies and microvascular thrombosis, it is administered as the activated enzyme, which has a short biological half-life. We have altered two putative inhibitory acidic residues near the thrombin cleavage site, which results in a 30-fold increase in substrate utilization by alpha-thrombin. We combined these changes with a genetically altered glycoform to generate a zymogen protein C with a 60-fold increased cleavage rate by free alpha-thrombin, independent of its cofactor thrombomodulin. We show that this 'proform' of protein C, unlike the natural circulating zymogen, can be activated by thrombin generated in clotting human plasma, resulting in an inhibition of further clot formation. Our data therefore show that we have engineered a site-activated agent, which only has anticoagulant activity when significant amounts of thrombin are being generated.     

Structural basis of cell surface receptor recognition by botulinum neurotoxin B

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.     

Distribution of factor VIII mRNA and antigen in human liver and other tissues

The cellular site of synthesis of factor VIII (FVIII:C; anti-haemophilic factor) has long been sought. Previous studies suggested the liver as a major site of synthesis, but extrahepatic sources such as spleen and lung have been implicated. Using an immunoradiometric assay (IRMA), we recently localized factor VIII antigen (FVIII:Ag, formerly FVIII:CAg), to whole perfused guinea pig liver and spleen, and to isolated hepatocytes, with lesser or trace amounts in other tissues. Using an immunohistological technique, Stel et al. detected FVIII:Ag in normal human liver sinusoidal endothelial cells, while Exner et al. detected FVIII:Ag by IRMA in extracts of human lymph nodes, lung, liver and spleen. The localization of antigen in tissues does not, however, distinguish sites of factor VIII synthesis from those of storage, and such experiments are subject to misinterpretation due to entrapment of plasma factor VIII in tissues. The recent cloning of the human factor VIII gene provides hybridization probes for the detection of factor VIII messenger RNA in cells, thus directly determining sites of synthesis. During complementary DNA cloning, we detected factor VIII mRNA in liver, and it has been localized by others in liver and placenta and in liver and kidney. In the present study, we detected factor VIII mRNA in isolated human hepatocytes, in spleen and in numerous tissues including lymph nodes and kidney, but not in white blood cells or cultured endothelial cells. We also found that the factor VIII, factor VII, factor IX and protein C antigens in liver are predominantly localized in hepatocytes, while very little von Willebrand factor antigen (vWF:Ag, formerly FVIIIR Ag) is detectable in this organ.     

The Clq receptor site on immunoglobulin G

We propose that, the binding site for the complement subcomponent Clq on immunoglobulin G involves the last two (C-terminal) beta-strands of the C gamma 2 domain. This region contains a large number of accessible and highly conserved charged residues and charge is postulated as an important component of the Clq-IgG interaction. The conclusions are reached on the basis of accessibility and sequence conservation analyses of C gamma 2 amino acid residues, the use of specific inhibitors and chemical modification studies.     

Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein

The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.     

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3, 4). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3-H4)(2) heterotetramer and are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.     

Crystal structure of the CorA Mg2+ transporter

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 A and 1.85 A resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+.     

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface.

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.     

Crystal structures of the membrane-binding C2 domain of human coagulation factor V

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.