Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Modulation of TRPV2 by endogenous and exogenous ligands: A computational study.

Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.

Structural insights into TRPV2 activation by small molecules.

Transient receptor potential vanilloid 2 (TRPV2) is involved in many critical physiological and pathophysiological processes, making it a promising drug target. Here we present cryo-electron microscopy (cryo-EM) structures of rat TRPV2 in lipid nanodiscs activated by 2-aminoethoxydiphenyl borate (2-APB) and propose a TRPV2-specific 2-ABP binding site at the interface of S5 of one monomer and the S4-S5 linker of the adjacent monomer. In silico docking and electrophysiological studies confirm the key role of His521 and Arg539 in 2-APB activation of TRPV2. Additionally, electrophysiological experiments show that the combination of 2-APB and cannabidiol has a synergetic effect on TRPV2 activation, and cryo-EM structures demonstrate that both drugs were able to bind simultaneously. Together, our cryo-EM structures represent multiple functional states of the channel, providing a native picture of TRPV2 activation by small molecules and a structural framework for the development of TRPV2-specific activators.

An Improved Substrate for Superior Imaging of Individual Biomacromolecules with Atomic Force Microscopy.

High-resolution atomic force microscopy (AFM) of biomacromolecules is a valuable method for structural studies in biology. Traditionally, the surfaces used for AFM imaging of individual molecules are limited to mica, graphite, and glass. Because these substrates have certain shortcomings, new or modified surfaces that improve the quality of AFM imaging are highly desirable. Here, we describe an improved substrate for imaging of individual biomacromolecules with high-resolution AFM based on graphite surfaces coated by physical adsorption. We provide a detailed methodology, including the chemical structure, synthesis, characterization and the use of a substance that modifies the surface of freshly cleaved graphite, making it suitable for adsorption and AFM visualization of various biomacromolecules while minimizing spatial distortions. We illustrate the advantages of the modified graphite over regular surfaces with examples of high-resolution single-molecule imaging of proteins, polysaccharides, DNA and DNA-protein complexes. The proposed methodology is easy to use and helps to improve substantially AFM imaging of biomacromolecules of various natures, including flexible and/or unstructured sub-molecular regions that are not seen on other AFM substrates. The proposed technique has the potential to improve the use of AFM in structural biology for visualization and morphometric characterization of macromolecular objects.

Factor XIII topology: organization of B subunits and changes with activation studied with single-molecule atomic force microscopy.

Essentials Factor XIII is a heterotetramer with 2 catalytic A subunits and 2 non-catalytic B subunits. Structure of active and inactive factor XIII was studied with atomic force microscopy. Inactive factor XIII is made of an A2 globule and 2 flexible B subunits extending from it. Activated factor XIII separates into a B2 homodimer and 2 monomeric active A subunits. SUMMARY: Background Factor XIII (FXIII) is a precursor of the blood plasma transglutaminase (FXIIIa) that is generated by thrombin and Ca2+ and covalently crosslinks fibrin to strengthen blood clots. Inactive plasma FXIII is a heterotetramer with two catalytic A subunits and two non-catalytic B subunits. Inactive A subunits have been characterized crystallographically, whereas the atomic structure of the entire FXIII and B subunits is unknown and the oligomerization state of activated A subunits remains controversial. Objectives Our goal was to characterize the (sub)molecular structure of inactive FXIII and changes upon activation. Methods Plasma FXIII, non-activated or activated with thrombin and Ca2+ , was studied by single-molecule atomic force microscopy. Additionally, recombinant separate A and B subunits were visualized and compared with their conformations and dimensions in FXIII and FXIIIa. Results and Conclusions We showed that heterotetrameric FXIII forms a globule composed of two catalytic A subunits with two flexible strands comprising individual non-catalytic B subunits that protrude on one side of the globule. Each strand corresponds to seven to eight out of 10 tandem repeats building each B subunit, called sushi domains. The remainder were not seen, presumably because they were tightly bound to the globular A2 dimer. Some FXIII molecules had one or no visible strands, suggesting dissociation of the B subunits from the globular core. After activation of FXIII with thrombin and Ca2+ , B subunits dissociated and formed B2 homodimers, whereas the activated globular A subunits dissociated into monomers. These results characterize the molecular organization of FXIII and changes with activation.

Polymorphism of G4 associates: from stacks to wires via interlocks.

We examined the assembly of DNA G-quadruplexes (G4s) into higher-order structures using atomic force microscopy, optical and electrophoretic methods, NMR spectroscopy and molecular modeling. Our results suggest that parallel blunt-ended G4s with single-nucleotide or modified loops may form different types of multimers, ranging from stacks of intramolecular structures and/or interlocked dimers and trimers to wires. Decreasing the annealing rate and increasing salt or oligonucleotide concentrations shifted the equilibrium from intramolecular G4s to higher-order structures. Control antiparallel and hybrid G4s demonstrated no polymorphism or aggregation in our experiments. The modification that mimics abasic sites (1',2'-dideoxyribose residues) in loops enhanced the oligomerization/multimerization of both the 2-tetrad and 3-tetrad G4 motifs. Our results shed light on the rules that govern G4 rearrangements. Gaining control over G4 folding enables the harnessing of the full potential of such structures for guided assembly of supramolecular DNA structures for nanotechnology.

Atomic Structural Models of Fibrin Oligomers.

The space-filling fibrin network is a major part of clots and thrombi formed in blood. Fibrin polymerization starts when fibrinogen, a plasma protein, is proteolytically converted to fibrin, which self-assembles to form double-stranded protofibrils. When reaching a critical length, these intermediate species aggregate laterally to transform into fibers arranged into branched fibrin network. We combined multiscale modeling in silico with atomic force microscopy (AFM) imaging to reconstruct complete atomic models of double-stranded fibrin protofibrils with γ-γ crosslinking, A:a and B:b knob-hole bonds, and αC regions-all important structural determinants not resolved crystallographically. Structures of fibrin oligomers and protofibrils containing up to 19 monomers were successfully validated by quantitative comparison with high-resolution AFM images. We characterized the protofibril twisting, bending, kinking, and reversibility of A:a knob-hole bonds, and calculated hydrodynamic parameters of fibrin oligomers. Atomic structures of protofibrils provide a basis to understand mechanisms of early stages of fibrin polymerization.

Thermal denaturation of fibrinogen visualized by single-molecule atomic force microscopy.

Fibrinogen denaturation is an important phenomenon in biology and medicine. It has been previously investigated with bulk methods and characterized by parameters, which refer to big protein ensembles. Here we provide a new insight into fibrinogen denaturation with a high-resolution single-molecule atomic force microscopy (AFM). The ultrastructure of individual fibrinogen molecules was studied after heating or extended contact with the highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM). Fibrinogen heating to 65 °C and 90 °C resulted in the formation of various shapes containing fibrillar and globular structures, which were attributed to the monomers and small aggregates of fibrinogen. Fibrinogen unfolded by the extended (10 min) incubation on GM-HOPG surface in water revealed a different morphology. It contained fibrillar structures only, and their organization reflected the initial native structure of fibrinogen: typically, six polypeptide chains connected by multiple disulfide bonds were seen. A combination of two morphologies - globular aggregates with dense fibrillar networks - has been revealed for thermally denatured protein adsorbed on a GM-HOPG surface with extended (10 min) rinsing with water. The obtained results provide better understanding of fibrinogen unfolding induced by different factors and are important for improvement of biomedical applications, such as fibrinogen-based protein matrixes and carbon-based biomaterials.

A coarse-grained model for DNA origami.

Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version.

Morphometric characterization of fibrinogen's αC regions and their role in fibrin self-assembly and molecular organization.

The flexible C-terminal parts of fibrinogen's Aα chains named the αC regions have been shown to play a role in fibrin self-assembly, although many aspects of their structure and functions remain unknown. To examine the involvement of the αC regions in the early stages of fibrin formation, we used high-resolution atomic force microscopy to image fibrinogen and oligomeric fibrin. Plasma-purified full-length human fibrinogen or des-αC fibrinogen lacking most of the αC regions, untreated or treated with thrombin, was imaged. Up to 80% of the potentially existing αC regions were visualized and quantified; they were highly heterogeneous in their length and configurations. Conversion of fibrinogen to fibrin was accompanied by an increase in the incidence and length of the αC regions as well as transitions from more compact conformations, such as a globule on a string, to extended and more flexible offshoots. Concurrent dynamic turbidimetry, confocal microscopy, and scanning electron microscopy revealed that trimming of the αC regions slowed down fibrin formation, which correlated with longer protofibrils, thinner fibers, and a denser network. No structural distinctions, except for the incidence of the αC regions, were revealed in the laterally aggregated protofibrils made of the full-length or des-αC fibrinogens, suggesting a pure kinetic effect of the αC regions on the fibrin architecture. This work provides a structural molecular basis for the promoting role of the αC regions in the early stages of fibrin self-assembly and reveals this stage of fibrin formation as a potential therapeutic target to modulate the structure and mechanical properties of blood clots.

Structural Basis of Interfacial Flexibility in Fibrin Oligomers.

Fibrin is a filamentous network made in blood to stem bleeding; it forms when fibrinogen is converted into fibrin monomers that self-associate into oligomers and then to polymers. To gather structural insights into fibrin formation and properties, we combined high-resolution atomic force microscopy of fibrin(ogen) oligomers and molecular modeling of crystal structures of fibrin(ogen) and its fragments. We provided a structural basis for the intermolecular flexibility of single-stranded fibrin(ogen) oligomers and identified a hinge region at the D:D inter-monomer junction. Following computational reconstruction of the missing portions, we recreated the full-atomic structure of double-stranded fibrin oligomers that was validated by quantitative comparison with the experimental images. We characterized previously unknown intermolecular binding contacts at the D:D and D:E:D interfaces, which drive oligomerization and reinforce the intra- and inter-strand connections in fibrin besides the known knob-hole bonds. The atomic models provide valuable insights into the submolecular mechanisms of fibrin polymerization.

The role of the 5'-cap structure in viral ribonucleoproteins assembly from potato virus X coat protein and RNAs.

The potato virus X (PVX) virion can be reconstituted in vitro from the virus coat protein (CP) and RNA; heterologous RNAs may be used as well. In our recent study, structure and properties of cognate and heterologous viral ribonucleoproteins (vRNPs) were demonstrated to be similar to those of native virions. The assembly was found to be initiated at the 5' terminus of an RNA and was not dependent on RNA sequence. The aim of the present study was to search for a signal or an essential structural element that directs packaging of viral genetic material into vRNPs. vRNPs were formed by incubation of the PVX CP with heterologous capped RNAs, their functional fragments lacking the cap structure, as well as the capped and uncapped transcripts corresponding to the 5'-terminal region of the genomic PVX RNA. Experimental data show that the presence of the cap structure at the 5' end of a nucleic acid is an important condition for vRNP assembly from RNA and CP. Presumably, the 5'-cap affects conformational state of the RNA region responsible for the efficient interaction with CP and creates conformational encapsidation signal for vRNP assembly.

Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type.

Investigation of early stages of fibrin association.

Interactions between fibrinogen molecules proteolytically cleaved with thrombin were investigated using atomic force microscopy (AFM) and dynamic light scattering (DLS). Gradually decreased fibrinogen concentrations were used to study the fibrin network, large separated fibrils, small fibrils in the initial association stages, and protofibrils. In addition, a new type of structure was found in AFM experiments at a low fibrinogen concentration (20 nM): the molecules in these single-stranded associates are arranged in a row, one after the other. The height, diameter, and distance between domains in these single-stranded associates were the same as those in the original fibrinogen molecules. DLS data assumed formation of extended associates in bulk solution at fibrinogen concentration as low as 20 nM.