Hexameric Assembly Research Articles

Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments.

Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell–cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.

Irreversible trimer to monomer transition of thermophilic rhodopsin upon thermal stimulation.

Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane α-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 °C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 °C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 °C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.

Solubility, Heat-Induced Gelation and Pepsin Susceptibility of Cruciferin Protein as Affected by Subunit Composition

This study examined the solubility, heat-induced gel formation ability, and pepsin susceptibility of cruciferin with mixed (wild type, WT) or identical subunits (CRUA, CRUB, or CRUC). All cruciferin species exhibited minimal solubility at pH 4.0 and slight solubility between pH 7.5 and 8, which was not influenced by the presence of NaCl. Increasing temperature to 90 °C did not affect the solubility of CRUC, but affected the solubility of other cruciferins between pH 4.0 and 8.0. Changes of secondary structure of cruciferins in a range of pH suggested structural alterations of the hexameric assembly might occur at pH 2.0. Near neutral pH was suitable to form heat-induced gels of WT, CRUA, and CRUB species. Although pH 2.0 was suitable for CRUC to form a strong gel, the same pH inhibited CRUB gel formation. High salt content (0.5 M NaCl) at neutral pH did not change the gel formation ability of WT, CRUA, or CRUB, but affected the gel characteristics. All cruciferins were hydrolyzed by pepsin, but the CRUC was degraded at a slower rate than the others. Trimer surface characteristics and the orientation and interaction of the HVR-I region of the hexameric assembly could be main structural factors for these property differences. Although the CRUC subunit showed properties that deviated from other species, the influence of CRUA and CRUB subunits seemed more prominent in the mixed subunit hexamer of WT.

Modular-DNA Programmed Molecular Construction of “Fixed” of 2D and 3D-Au Nanoparticle Arrays

Specifically designed dimeric, planar trimeric, tetrameric, pentameric, and 3D hexameric assemblies of Au nanoparticles were constructed from self-assembling DNA modules that contain disulfide groups, for attachment to the nanoparticles, and covalently linked 2,5-bis(2-thienyl)pyrrole (SNS) monomers. Treatment of these arrays with horseradish peroxidase and H2O2 (HRP/H2O2) results in bond formation between the SNS monomers that cross-links or ligates the DNA modules making the assemblies permanent (“fixed”).

AccD1 And AccA1 from M. tuberculosis form A dodecameric MCC-type holo complex

Mycobacteria have an unusual redundancy of six putative carboxyltransferase genes that form high-molecular weight holo acyl coenzyme A carboxylase complexes with a complementary set of three biotin carboxylase genes. Most of these enzyme complexes use small fatty acid coenzyme A esters as substrate, to allow their extension by one methylene group via a carboxybiotin-mediated α-carboxylation reaction. Redundant occurrence of these complexes was assumed to be related to highly complex enzymatic requirements in lipid biosynthesis, as the mycobacterial thick cell wall comprises unusual very long chain fatty acids, including mycolic acid. We have solved two high-resolution crystal structures of the 350 kDa hexameric assemblies of two different acyl coenzyme A carboxylase hexameric assemblies, AccD5 and AccD6 [1; Anandhakrishnan et al., unpublished], and characterized these enzyme complexes functionally. In a second step we investigated the acyl coenzyme A carboxylase complex AccD1-AccA1 from Mycobacteria tuberculosis with hitherto unknown function. By using a metabolomics approach we found that AccD1-AccA1 is involved in branched amino acid catabolism, which was not investigated in mycobacteria before [Ehebauer et al, unpublished. Using an in vitro assay, we show that the enzyme complex uses methylcrotonyl coenzyme A as substrate]. We determined the overall architecture of the 700 kDa AccD1-AccA1 complex to be formed from three layers of a central AccD1 hexameric ring, flanked by two distal tiers composed of three AccA1 subunits each. Our electron microscopy data match the overall dimensions of a methylcrotonyl coenzyme A holo complex with known structure and thus support our functional findings. Our data suggest a unique functional role of the AccD1-AccA1 complex within the Mycobacterium tuberculosis acyl coenzyme A carboxylase interactome. Ultimately, it is our goal to solve this and related structures of ACCase holo complexes by high-resolution crystallography as well. The abstract is dedicated to Louis Delbaere with whom I shared time during my PhD at the University of Basel, Switzerland.

Native Hexameric Full-Length HIV-1 Capsid: Crystal Structure and Drug Targeting

The HIV-1 full length capsid protein (CA-FL) is increasingly viewed as an attractive therapeutic target since proper capsid formation is required for viral infection. CA-FL is synthesized as a central domain of a structural Gag polyprotein that is involved in both early and late stages of the viral life cycle. During the HIV-1 maturation process, Gag is cleaved by a viral protease to produce several discrete new proteins that include matrix, capsid (CA-FL), and nucleocapsid. After proteolytic cleavage, CA-FL forms hexamers and pentamers that rearrange into a fullerene cone-shaped structure, which surrounds the viral genome at the center of the mature virus. Crystal structures of the native unassembled hexameric CA-FL (without cross-linked residues that might prevent changes in the inter- or intra-subunit interactions) are of great interest, as they may provide insights relevant to the development of drugs that prevent or impede the transition from the preassembled to the assembled capsid states. Recently, we crystallized and solved the crystal structure of the first hexameric HIV-1 CA-FL in its native form (without engineered cross-linking cysteines). There is one molecule per asymmetric unit, and the P6 space group generates the native hexameric assembly. We have also identified a small molecule, 18E8, which exhibits broad anti-HIV activity in cell-based assays, and targets CA-FL. This was demonstrated by experiments that selected for viruses with drug resistance and revealed that an A105T mutation in CA-FL confers resistance to the compound. Time-of-inhibitor addition experiments showed that 18E8 targets an early step in the HIV replication cycle, after reverse transcription and before integration. Electron microscopy experiments suggest that 18E8 does not impart significant morphological changes in CA-FL tubular assemblies. Our structure of CA-FL and our ongoing work with the CA-FL/18E8 complex will provide a system for the investigation of molecular interactions between CA-FL and small molecule antivirals that work with a novel mechanism of action.

Structure of tomato wound-induced leucine aminopeptidase sheds light on substrate specificity.

The acidic leucine aminopeptidase (LAP-A) from tomato is induced in response to wounding and insect feeding. Although LAP-A shows in vitro peptidase activity towards peptides and peptide analogs, it is not clear what kind of substrates LAP-A hydrolyzes in vivo. In the current study, the crystal structure of LAP-A was determined to 2.20 Å resolution. Like other LAPs in the M17 peptidase family, LAP-A is a dimer of trimers containing six monomers of bilobal structure. Each monomer contains two metal ions bridged by a water or a hydroxyl ion at the active site. Modeling of different peptides or peptide analogs in the active site of LAP-A reveals a spacious substrate-binding channel that can bind peptides of five or fewer residues with few geometric restrictions. The sequence specificity of the bound peptide is likely to be selected by the structural and chemical restrictions on the amino acid at the P1 and P1'positions because these two amino acids have to bind perfectly at the active site for hydrolysis of the first peptide bond to occur. The hexameric assembly results in the merger of the open ends of the six substrate-binding channels from the LAP-A monomers to form a spacious central cavity allowing the hexameric LAP-A enzyme to simultaneously hydrolyze six peptides containing up to six amino acids each. The hexameric LAP-A enzyme may also hydrolyze long peptides or proteins if only one such substrate is bound to the hexamer because the substrate can extend through the central cavity and the two major solvent channels between the two LAP-A trimers.

Polymorphs of 1-(5-Methylthiazol-2-yl)-3-phenylthiourea and Various Anion-Assisted Assemblies of Two Positional Isomers

Orientations of the phenyl group and the intramolecular hydrogen bond play prime roles in the packing patterns of three conformational polymorphs of an unsymmetrical thiourea derivative, 1-(5-methylthiazol-2-yl)-3-phenylthiourea (PTH1). Self-assembly of each polymorph is composed of hydrogen-bonded dimeric motifs held together in head-to-tail arrangement but packed in different manners. Each has an intramolecular N–H···N hydrogen bond between an amide N–H and the nitrogen atom of the 5-methylthiazole unit. The packing pattern of 1-(4-methylthiazol-2-yl)-3-phenylthiourea (PTH2) is composed of dimeric assemblies of PTH2 in head-to-tail fashion. PTH2 is monomorphic as there are intermolecular C–H···S interactions between a C–H bond of the phenyl ring of each molecule and the sulfur atom of the thiocarbonyl group of a neighboring molecule. Such interactions lock the orientation of phenyl group in the solid state. The syn–anti conformation across the thiourea group, originally present in the positional isomers PTH1 and PTH2, is invariably transformed to syn–syn conformation in their salts. The extent of hydration of anions in the salts of PTH1 or PTH2 is dependent on the cation as well as the anion. The chloride salt of PTH1 has a large difference in packing patterns in comparison with the corresponding chloride salt of PTH2; they also differ in the numbers of symmetry-nonequivalent molecules in their respective unit cells. The anhydrous salt of PTH1 with hydrogen bromide having a 1:1 ratio of cation and anion is formed, whereas the bromide salt of PTH2 is a hydrate of composition (HPTH2)2(Br)2·6H2O. This salt has bromide–water clusters in its crystal lattice. Nitric acid reacts with PTH1 under different conditions to form hydrated or anhydrous salts. The hydrated salt (HPTH1)2(NO3)2·H2O has anions bridged by water molecules. The anhydrous nitrate salts of PTH1 and PTH2 are structurally similar in having nitrate···nitrate interactions. The deprotonation of polyacids by PTH1 and PTH2 is selective. The ability to abstract a proton from sulfuric acid to form crystalline salts by PTH1 and PTH2 differs. The sulfate salt (HPTH1)2(SO4) is formed by reaction of sulfuric acid with PTH1, but PTH2 forms the bisulfate salt (HPTH2)HSO4·H2O. PTH1 forms the corresponding dihydrogen phosphate salt upon reaction with orthophosphoric acid; the dihydrogen phosphate anions are held together in the form of cyclic hydrogen-bonded hexameric assemblies in the lattice. Water loss from the assemblies of hydrated salt was determined by thermogravimetry and differential scanning calorimetry and showed that dehydration from anion-assisted assemblies was guided by the cationic host and the type of assembly.

Connexins in Renal Endothelial Function and Dysfunction

The renal endothelium plays a critical role in kidney physiopathology as it is implicated in various processes such as the regulation of vasomotor tone, the control of tissue inflammation and thrombosis. Recent evidence highlights direct implication of renal endothelial dysfunction in the progression of chronic kidney disease. Renal endothelial dysfunction is a multifaceted process in which chemokines, cytokines, prothrombotic factors and adhesion molecules are known to play a crucial role. Apart from paracrine cell-to-cell signaling, the role for gap junction-mediated intercellular communication in renal physiopathology has been recently suggested. Gap junction channels are formed by the hexameric assembly of connexins and directly connect the cytoplasm of adjacent cells. Due to their ability to regulate multiple physiological and pathological signals connexins are currently taking an important place in the list of actors involved in renal endothelial function and dysfunction. In this review we will focus on possible implications of connexins in the physiopathological processes associated with renal vascular endothelium.

Exploring Dynamic Events of Bacterial Microcompartment Shell Pores

Bacterial Microcompartments (BMCs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency and prevent the loss of volatile or cytotoxic intermediates. 7 distinct BMC-type operons have been identified in 20-25% of bacteria, including BMCs involved in carbon fixation, propanediol catabolism, and ethanolamine catabolism. Ranging from 100 to 150 nm in width, BMCs are encapsulated in a protein shell consisting of hexagonal tiles of the conserved BMC-fold proteins. The BMC-fold proteins homo-oligomerize into hexameric or pseudo-hexameric assemblies with unique functionalities. High resolution X-ray determined structures have previously elucidated the organization and tiling interactions of several shell proteins from BMCs. However, it remains unknown how small molecule metabolites may enter or exit the BMC, passing through the protein shell; nor how intermediates may be trapped inside. Here, we present initial findings using current methods in Molecular Dynamics to investigate BMC shell pores, including: (1) Metadynamics and Umbrella Sampling to probe the free energy profile of small molecule metabolites at predicted routes through BMC shell protein hexamer pores; (2) Accelerated MD and Targeted MD to observe the conformational change between BMC shell protein pseudo-hexamers that have been solved in open and closed pore conformations. These experiments shed insight on BMC shell pore dynamics and function, furthering our understanding of BMC systems.

Mechanisms of Molecular Transport through the Proton-Gated Urea Channel of Helicobacter Pylori

Helicobacter pylori survival in acidic environments relies on cytoplasmic hydrolysis of gastric urea into ammonia and carbon dioxide, which buffer the pathogen's periplasm. Urea uptake is greatly enhanced and regulated by HpUreI, a proton-gated inner-membrane channel protein essential for gastric survival of H. pylori. The crystal structure of HpUreI describes a static snapshot of the channel with two constriction sites near the center of the bilayer that are too narrow to allow passage of urea or even water. Here we report the atomic resolution urea transport mechanism, revealed by unrestrained microsecond equilibrium molecular dynamics simulations of the hexameric channel assembly. Two consecutive constrictions open to allow conduction of urea, which is guided through the channel by an interplay of conserved residues that determine proton rejection and solute selectivity. Remarkably, HpUreI conducts water at rates equivalent to aquaporins, which might be essential for efficient transport of urea at small concentration gradients.

Structural Modeling of Hexameric and Tetrameric Ion Conduction Pathways of Orai1 Channel

Ca2+ release-activated Ca2+ (CRAC) channels mediate Ca2+ entry in response to store depletion in a variety of cell types. Endogenous CRAC channels in mammals are formed by a homomeric assembly of Orai1 proteins. Earlier functional studies involving chemical cross-linking of different numbers of wild-type Orai1 subunits, electrophysiological recordings and single molecule fluorescence analysis techniques as well as high-resolution electron microscopic examination of purified Orai proteins strongly indicated that functional Orai channels were most likely tetramers. In contrast, study of crystals containing holo-dOrai channel complexes from Drosophila melanogaster, revealed that a single channel complex contains six dOrai subunits. The hexameric CRAC channel stoichiometry was further supported by cross-linking and size-exclusion chromatography studies of the Drosophila Orai. Recent functional study revealed that expression of concatenated hexameric Orai1 channel produces a non-selective cation channel with biophysical properties essentially different from those of endogenous CRAC channels or channels formed by expressed concatenated tetrameric Orai1 proteins. Thus, the studies reported are far from conclusive but they do indicate the need for further investigation of Orai1 channel stoichiometry. Accordingly, we generated structural models of Orai1 ion conduction pathway formed by tetrameric or hexameric assembly of Orai1 pore-lining TM1 segments using Rosetta fold and dock protocol. Based on available experimental data we constrained proximity of several key residues lining Orai1 ion conduction pathway during simulations. Among the lowest energy and most frequently sampled conformations of Orai TM1 hexamers generated by Rosetta, we identified structural models that were in close agreement with Orai structure in the transmembrane region proposed based on crystallographic data analysis. Our preliminary models of Orai TM1 tetramers suggest alternative structural topology forming ion conduction pathway, which may account for the differences in ionic selectivity of hexameric and tetrameric Orai1 channels.

Mechanisms of molecular transport through the urea channel of Helicobacter pylori

Helicobacter pylori survival in acidic environments relies on cytoplasmic hydrolysis of gastric urea into ammonia and carbon dioxide, which buffer the pathogen’s periplasm. Urea uptake is greatly enhanced and regulated by HpUreI, a proton-gated inner membrane channel protein essential for gastric survival of H. pylori. The crystal structure of HpUreI describes a static snapshot of the channel with two constriction sites near the center of the bilayer that are too narrow to allow passage of urea or even water. Here we describe the urea transport mechanism at atomic resolution, revealed by unrestrained microsecond equilibrium molecular dynamics simulations of the hexameric channel assembly. Two consecutive constrictions open to allow conduction of urea, which is guided through the channel by interplay between conserved residues that determine proton rejection and solute selectivity. Remarkably, HpUreI conducts water at rates equivalent to aquaporins, which might be essential for efficient transport of urea at small concentration gradients.

Enhanced IgG Hexamerization Mediates Efficient C1q Docking and Complement-Dependent Cytotoxicity; Preclinical Proof Of Concept On Primary CLL and Burkitt Lymphoma

IntroductionWe recently discovered that complement is activated by IgG hexamers that assemble on the surface of cells following antigen binding. The formation of hexamers is dependent on intermolecular interactions in the Fc interface between neighboring IgG molecules. This seminal finding allowed us to identify mutations that enhance the ability of IgG antibodies to bind C1 and induce complement-dependent lysis. ObjectivesTo study the role of Fc-mediated intermolecular contacts in complement-dependent cytotoxicity (CDC) of cancer cells by therapeutic IgG antibodies in biological relevant in vitro and in vivo model systems and to exploit this mechanism to improve CDC activity. M&MBased on an IgG hexamer crystal structure, we generated Fc mutations that were predicted to influence Fc:Fc interactions between IgG antibodies and we screened for improved C' activation. The effect of these mutations on in vitro C1q binding and CDC-mediated killing of tumor B cell lines and patient-derived chronic lymphatic leukemia (CLL) cells was studied with therapeutic antibodies targeting different B cell antigens. The effect of a CDC-enhancing mutation was further studied in a Burkitt lymphoma xenograft model. ResultsHere we show that the assembly of IgG hexamers on the cell surface represents an important step for efficient complement activation via the classical pathway by therapeutic antibodies against hematological targets. Critical amino acid residues in the Fc:Fc contacts between IgG molecules were identified and individual mutants with increased complement activation generated. Interestingly, introduction of an enhancing mutation was shown to significantly increase complement activation not only of antibodies capable of inducing CDC, but also of antibodies that were devoid of such activity. We provide a proof-of-concept for CD19, Type I and Type II CD20, CD38 and CD52 antibodies. Importantly, we were able to achieve or enable significant CDC of a panel of primary CLL cells by use of this novel approach. Finally, we show that the enhancing mutation resulted in a significant increase in the inhibition of in vivo tumor growth by a CD20 antibody in a subcutaneous Raji xenograft model in SCID mice. ConclusionThe data obtained provide a novel insight into the mechanism of complement-dependent lysis. This mechanism is shown to be relevant for a panel of therapeutic antibodies against hematological targets. Mutations that improved the formation of Fc:Fc-mediated hexamerization resulted in strongly increased CDC of complement-resistant primary CLL cells and improved efficacy in an in vivo model with a Burkitt lymphoma cell line. Disclosures:Beurskens:Genmab: Employment. de Jong:Genmab: Employment. Verploegen:Genmab: Employment. Voorhorst:Genmab: Employment. Strumane:Genmab: Employment. Lindorfer:Genmab: Research Funding. Taylor:Genmab: Research Funding. Schuurman:Genmab: Employment. Parren:Genmab: Employment.

FTIR analysis of the high pressure response of native insulin assemblies

It is widely recognized that a central role in conferring stability to the structure of proteins against misfolding and aggregation is played by the formation of oligomers. The case of insulin is prototypical in this respect: in our body it is stored up in stable inactive hexameric assemblies whereas only in its monomeric form it recovers the role of regulating carbohydrate and fat metabolism. In the present paper, exploiting the optimal coupling between FTIR spectroscopy and diamond anvil cell technique, we probe the stability of different insulin oligomeric forms under high pressure, namely over the ranges 0–15kbar for water solution and 0–80kbar for dry powder. Results obtained show different responses to volume compression for the different assemblies being the structure of monomers and dimers remarkably more affected by compression than hexamers. Moreover by comparing the results obtained using water solution and dry powder we were able to draw important considerations about the role of water in the high pressure unfolding processes.

Unusual architecture of the p7 channel from hepatitis C virus

The Hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations1-4. We are curious as to what structural solution has the viroporin adopted to afford selective cation conduction because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The p7 activity can be inhibited by amantadine and rimantadine2,5, which also happen to be potent blockers of the influenza M2 channel6 and licensed drugs against influenza infections7. The adamantane derivatives were subjects of HCV clinical trials8, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here, we determined the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbors, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also alludes to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter while an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording showed that these residues are indeed critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Supramolecular Approaches to Combining Membrane Transport with Adhesion

Cells carefully control the transit of compounds through their membranes using “gated” protein channels that respond to chemical stimuli. Connexin gap junctions, which are high conductance cell-to-cell channels, are a remarkable class of “gated” channel with multiple levels of assembly. A gap junction between adhering cells comprises two half-channels in each cell membrane that adhere to each other to form a continuous cell-to-cell channel. Each half-channel is a hexameric assembly of six protein transmembrane subunits. These gap junctions display both intramembrane assembly and intermembrane assembly, making them an attractive target for biomimetic studies. Although many examples of self-assembled channels have been developed, few can also mediate intermembrane adhesion. Developing systems that combine membrane adhesion with controlled transit across the membrane would not only provide a better understanding of self-assembly in and around the membrane, but would also provide a route towards smart biomaterials, targeted drug delivery and an interface with nanotechnology.This Account describes our biomimetic approaches to combining membrane adhesion with membrane transport, using both self-assembled “sticky” pores and “sticky” nanoparticles to trigger transit across membranes. This combination links both fundamental and applied research, acting as a bridge between molecular level assembly and the formation of functional biomaterials. The ultimate goal is to create complex self-assembled systems in biological or biomimetic environments that can both interface with cells and transport compounds across bilayers in response to remote chemical or electromagnetic signals. Our research in this area started with fundamental studies of intramembrane and intermembrane self-assembly, building upon previously known channel-forming compounds to create self-assembled channels that were switchable or able to mediate vesicle–vesicle adhesion. Subsequently, nanoparticles with a “sticky” coating were used to mediate adhesion between vesicles. Combining these adhesive properties with the unique characteristics of nanosized magnetite allowed a noninvasive magnetic signal to trigger transport of compounds out of magnetic nanoparticle-vesicle assemblies. Adding an extravesicular matrix produced new responsive biomaterials for use in tissue engineering. These biomaterials can be magnetically patterned and can deliver drugs upon receipt of a magnetic signal, allowing spatiotemporal control over cellular responses.

Structural Insights into the UbiD Protein Family from the Crystal Structure of PA0254 from Pseudomonas aeruginosa

The 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) catalyzes the conversion of 3-polyprenyl-4-hydroxybenzoate to 2-polyprenylphenol in the biosynthesis of ubiquinone. Pseudomonas aeruginosa contains two genes (PA0254 and PA5237) that are related in sequence to putative UbiD enzymes. A bioinformatics analysis suggests that the UbiD sequence family can be divided into two subclasses, with PA5237 and PA0254 belonging to different branches of this family. The three-dimensional structure of PA0254 has been determined using single wavelength anomalous diffraction and molecular replacement in two different crystal forms to resolutions of 1.95 and 2.3 Å, respectively. The subunit of PA0254 consists of three domains, an N-terminal α/β domain, a split β-barrel with a similar fold of a family of flavin reductases and a C-terminal α/β domain with a topology characteristic for the UbiD protein family. The middle domain contains a metal binding site adjacent to a large open cleft that may represent the active site. The two protein ligands binding a magnesium ion, His188 and Glu229, invariant in the PA0254 subclass, are also conserved in a corresponding metal site found in one of the FMN binding proteins from the split β-barrel fold family. PA0254 forms, in contrast to the hexameric UbiD from E. coli and P. aeruginosa, a homo-dimer. Insertion of four residues in a loop region in the PA0254 type enzymes results in structural differences that are incompatible with hexamer assembly.

Purification and Biophysical Characterization of an 11S Globulin from Wrightia tinctoria Exhibiting Hemagglutinating Activity

Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS-PAGE gel under non-reducing conditions, a major band of ~56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ~32-34 kDa and the other with molecular weight ~22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of β-sheets (44.2%) and moderate content of α-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 Å°C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.

Insights into FlaI Functions in Archaeal Motor Assembly and Motility from Structures, Conformations, and Genetics

Superfamily ATPases in type IV pili, type 2 secretion, and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here, we structurally and functionally characterize prototypical superfamily ATPase FlaI in Sulfolobus acidocaldarius, showing FlaI activities in archaeal swimming-organelle assembly and movement. X-ray scattering data of FlaI in solution and crystal structures with and without nucleotide reveala hexameric crown assembly with key cross-subunit interactions. Rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other and distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility, and force direction via a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify invivo functional components for assembly and motility, phosphate-triggered rearrangements by ATP hydrolysis, and molecular predictors for distinct ATPase superfamily functions.