Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

University of Texas Medical Branch

university of texas medical branch

Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo-3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm(2). Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex. (Blood. 2002;99:1215-1223)

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography.

A recently identified class of proteins conferring insecticidal activity to several bacteria within the Enterobacteriaceae family have potential for control of commercially important insect pests. Here, we report the first purification, biophysical characterisation and 3-D structural analysis of one of the toxin components, XptA1, from Xenorhabdus nematophila PMFI296 to a resolution of 23 A. Membrane binding studies indicate that the three-component toxin system has a different mode of action from that of proteins from Bacillus thuringiensis (Bt). Biophysical characterisation of XptA1 suggests a mechanism of action of XptA1 whereby it first binds to the cell membrane forming a structure with a central cavity and forms a complex with its partners XptB1 and XptC1 producing the full insecticidal toxin. The structure of XptA1 is shown by a combination of electron microscopy, ultracentrifugation and circular dichroism spectroscopy to be a 1.15 MDa tetramer with a cage-like structure. Each of the four symmetry-related subunits has three well-defined domains and a longitudinal twist with one end narrower than the other. One third of the residues of XptA1 are alpha-helical and it is suggested the subunits associate partly via an alpha-helical coiled-coil interaction. XptA1 itself shows the same secondary structure at neutral pH and in an alkaline environment up to pH10.5. This pH tolerance indicates that the folded XptA1 can pass through the midgut of Lepidopteran insects susceptible to the insecticidal toxin complex. This implies therefore that its folded structure is important for its biological activity.

Structural characterisation of the insecticidal toxin XptA1, reveals a 1.15 MDa tetramer with a cage-like structure.

Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.

Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes.

Cryo-electron microscopy has the power to visualise lipid membranes at the closest to in vivo conditions. The structure of the lipid bilayer can be well resolved and the interactions between lipid-protein and protein-protein molecules followed at the molecular level. We undertook an extended Cryo-electron microscopy study to follow the factor VIII binding to phosphatidylserine containing lipid nanotubes at different lipid composition. Obtaining well ordered tubes is required to define the factor VIII membrane-bound structure. The observed alterations in the arrangement of the protein molecules are indicative for the flexibility of the membrane-bound factor VIII. Understanding the significance of these conformational changes is essential to comprehend the function of factor VIII in coagulation and as a drug for Hemophilia A.

Binding of recombinant human coagulation factor VIII to lipid nanotubes.

In this study, an MS-based proteomics approach to characterizing the virion structural proteins of the novel marine 'photosynthetic' phage S-PM2 is presented. The virus infects ecologically important cyanobacteria of the genus Synechococcus that make a substantial contribution to primary production in the oceans. The S-PM2 genome encodes 236 ORFs, some of which exhibit similarity to known phage virion structural proteins, but the majority (54%) show no detectable homology to known proteins from other organisms. Using public and in-house bioinformatics tools the proteome of S-PM2 was predicted and a database compatible with MS-based search engines was constructed. S-PM2 virion proteins were resolved by SDS-PAGE, excised, tryptically digested and analysed by LC-ESI-MS/MS. The resulting MS data were searched against the database. A parallel control study was undertaken on the well-characterized coliphage T4 in order to assess the sensitivity and efficiency of this approach. In total, 11 of the 15 S-PM2 proteins, predicted to be virion proteins by bioinformatics approaches, were confirmed as such, together with the identification of a further 12 novel structural proteins. In the case of T4, 24 of the 39 known virion structural proteins were identified, including the major tail-fibre proteins. This approach has wide-ranging applicability and can be applied to any novel organism whose genome encodes ORFs with few detectable homologies in the public databases.

A proteomic approach to the identification of the major virion structural proteins of the marine cyanomyovirus S-PM2.

Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg(63), Lys(70), Lys(101), Glu(138), Asp(139), and Asn(160) engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.

Engineered annexin A5 variants have impaired cell entry for molecular imaging of apoptosis using pretargeting strategies.

In this review article the theoretical and experimental possibilities of applying EO-methods for estimation of the physico-chemical properties of the organic nanotubes (ONTs) are studied. The ONTs are highly organized nanostructures of strongly elongated, anysometric, and hollow cylinders with a size range of 1 nm to 10,000 nm, e.g. in aqueous solutions they could behave as colloid (disperse) particles. They have high interaction ability due to their extremely large curved, rolled-up external surfaces (bilayers of membrane walls) and unique properties because of their specific electric charge distribution and dynamics that make possible the functionalization of their surfaces. Thus they could template guestsubstances such as membrane proteins and protein complexes on the exterior surfaces and in the membrane. We performed our investigations for the case of ONT aqueous colloid suspension. Following our earlier proposition of the general expression for the electro-optic (EO) effect we derived equations for the evaluation of the electric properties of ONT particles such as mechanism of electric polarization and identification of their most important electric Dipole Moments (DM), permanent (pDM) and induced (iDMs). Further we recommend ways for the calculation of their magnitude and direction. Also we evaluated some geometrical properties such as length of the ONT particles and their polydispersity. The knowledge that we provided about the ONT properties may enable us to elucidate and predict their biological activity. Templating biological active ligands (such as membrane proteins and protein complexes) on the inner and outer surfaces as well as in the surface membrane creates their potential usefulness as carrier and deliverer of biopharmaceuticals in bio-nanodevices. The theoretical equations were compared with the experimental data for ONTs such as (lipid) LNT, Tobacco Mosaic Virus (TMV) and microtubules (MT). Comparison of EO methods with other methods used till now shows that the EO methods are faster, not invasive and do not alter the studied particles.

Electro-optic properties of organic nanotubes.

Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.

Bioluminescence methodology for the detection of protein-protein interactions within the voltage-gated sodium channel macromolecular complex.

Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 448-459, 2013.

Domain organization of membrane-bound factor VIII.

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.

Svetla Stoilova-McPhie

Department of Neuroscience and Cell Biology,
Sealy Center for Structural Biology and Molecular Biophysics

University of Texas Medical Branch

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Svetla Stoilova-McPhie

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neuroscience and Cell Biology,Sealy Center for Structural Biology and Molecular Biophysics

University of Texas Medical Branch

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Department of Neuroscience and Cell Biology,
Sealy Center for Structural Biology and Molecular Biophysics