Kirill Grushin

Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission

Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 ‘MUN’ domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work‐bench on which SNAREpins are templated. This ‘buttressed‐ring hypothesis’ affords straightforward answers to many principal and long‐standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential.

Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII.

Factor VIII (FVIII) is a multidomain blood plasma glycoprotein. Activated FVIII acts as a cofactor to the serine protease factor IXa within the membrane‐bound tenase complex assembled on the activated platelet surface. Defect or deficiency in FVIII causes haemophilia A, a severe hereditary bleeding disorder. Intravenous administration of plasma‐derived FVIII or recombinant FVIII concentrates restores normal coagulation in haemophilia A patients and is used as an effective therapy. In this work, we studied the biophysical properties of clinically potent recombinant FVIII forms: human FVIII full‐length (FVIII‐FL), human FVIII B‐domain deleted (FVIII‐BDD) and porcine FVIII‐BDD bound to negatively charged phospholipid vesicles at near‐physiological conditions. We used cryo‐electron microscopy (Cryo‐EM) as a direct method to evaluate the homogeneity and micro‐organization of the protein‐vesicle suspensions, which are important for FVIII therapeutic properties. Applying concurrent Cryo‐EM, circular dichroism and dynamic light scattering studies to the three recombinant FVIII forms when bound to phospholipid vesicles revealed novel properties for their functional, membrane‐bound state. The three FVIII constructs have similar activity, secondary structure distribution and bind specifically to negatively charged phospholipid membranes. Human and porcine FVIII‐BDD induce strong aggregation of the vesicles, but the human FVIII‐FL form does not. The proposed methodology is effective in characterizing and identifying differences in therapeutic recombinant FVIII membrane‐bound forms near physiological conditions, because protein‐containing aggregates are considered to be a factor in increasing the immunogenicity of protein therapeutics. This will provide better characterization and development of safer and more effective FVIII products with implications for haemophilia A treatment.

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

Factor VIII organisation on nanodiscs with different lipid composition

Nanodiscs (ND) are lipid bilayer membrane patches held by amphiphilic scaffolding proteins (MSP) of ~10 nm in diameter. Nanodiscs have been developed as lipid nanoplatforms for structural and functional studies of membrane and membrane associated proteins. Their size and monodispersity have rendered them unique for electron microscopy (EM) and single particle analysis studies of proteins and complexes either spanning or associated to the ND membrane. Binding of blood coagulation factors and complexes, such as the Factor VIII (FVIII) and the Factor VIIIa - Factor IXa (intrinsic tenase) complex to the negatively charged activated platelet membrane is required for normal haemostasis. In this study we present our work on optimising ND, specifically designed to bind FVIII at close to physiological conditions. The binding of FVIII to the negatively charged ND rich in phosphatidylserine (PS) was followed by electron microscopy at three different PS compositions and two different membrane scaffolding protein (MSP1D1) to lipid ratios. Our results show that the ND with highest PS content (80 %) and lowest MSP1D1 to lipid ratio (1:47) are the most suitable for structure determination of the membrane-bound FVIII by single particle EM. Our preliminary FVIII 3D reconstruction as bound to PS containing ND demonstrates the suitability of the optimised ND for structural studies by EM. Further assembly of the activated FVIII form (FVIIIa) and the whole FVIIIa-FIXa complex on ND, followed by EM and single particle reconstruction will help to identify the protein-protein and protein-membrane interfaces critical for the intrinsic tenase complex assembly and function.

Lipid nanotechnologies for structural studies of membrane-associated proteins

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane‐associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo‐electron microscopy (cryo‐EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self‐assemble in aqueous solutions. The lipid nanodisks (NDs) are self‐assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane‐associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelets phosphtaidylserine rich membrane and have successfully assembled functional membrane‐bound coagulation factor VIII in vitro for structure determination by cryo‐EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three‐dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane‐associated proteins and complexes for structural studies by cryo‐EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane‐associated proteins, such as the coagulation factors, at a close to physiological environment. Proteins 2014; 82:2902–2909.

Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5)

The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/Ls ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21−49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.

Reversible stacking of lipid nanodiscs for structural studies of clotting factors

Abstract Nanodiscs (ND) are discoidal phospholipid bilayers stabilized by a pair of membrane-scaffolding proteins (MSP). The macromolecular composition and size of ND are ideal for structural and functional studies of membrane and membrane-associated proteins. In this work, we investigate the assembly of ND from a galactosylceramide and dioleoyl phosphatidylserine (PS) lipid mixture with two different MSP and at four MSP-to-lipid ratios. This lipid composition has been optimized for structural and biophysical studies of membrane-bound blood clotting factors that require Ca2+ ions for function. We have demonstrated that CaCl2 induces reversible stacking of the ND that depends on the ND size and Ca2+ concentrations. Our biophysical and electron microscopy (EM) studies show a predominant ND population of ~12 nm in diameter for both the ND assembled from MSP1D1 to lipids ratio of 1:40 and from MSP1E3D1-to-lipids ratio of 1:80. Approximately half of the ND population assembled at MSP1E3D1-to-lipids ratio of 1:150 has a diameter of ~16 nm. These larger ND form ordered stacks at 5-mm Ca2+ concentrations, as shown by cryo-EM. The number and length of the ND stacks increase with the increasing in Ca2+ concentration. Adding millimolar concentrations of EDTA reverses the stacking of the ND.

L-arginine Decreases L-type Ca2+ Current Through Receptor Activation Of NO-cGMP Cascade. Enigma Of “Arginine Paradox”

One of the most important problems, related to synthesis of NO, is the problem of “arginine paradox”. In preliminary studies we demonstrated, that arginine paradox is realized not only in endothelial cells, but also in isolated cardiomyocytes. The aim of this study was to investigate receptor hypothesis of “arginine paradox” formation in isolated rat cardiomyocytes. Thus we studied the regulation of the basal L-type Ca2+ current by NO-cGMP cascade using the patch clamp method.In the presence of 1 mM L-arginine in all experimental media the activation of NO-cGMP cascade by 5 mM L-arginine induced a steady suppression of L-type Ca2+ current amplitude on average by 30% in all experiments, which has not been observed previously. In the presence of 1 μM 7NI (NOS blocker) or KT5823 (PKG blocker), addition of 5 mM arginine had no marked effect on the amplitude of L-type Ca2+ current.Next we investigated whether addition of 5 mM L-arginine affects the activation of α2-adrenoceptors. It was shown that in the presence of α2-adrenoceptor antagonists - yohimbine (10 μM) and rauwolscine (10 μM), 5 mM of arginine had no effect on the amplitude of L-type Ca2+ current. Then activity of some key components of the cascade activating NO synthase through α2-adrenoceptors was checked. We showed that in the presence of 100 nM wortmannin (PIP3 kinase blocker) or 0,5 μM Akt1/2 inhibitor (PKB bloker) 5 mM of L-arginine had no marked effect on L-type Ca2+ current.Thus we have shown that extra addition of L-arginine affects the amplitude of L-type Ca2+ current and related with the activation of α2-adrenoceptors followed by an increase in NOS activity.