Helix Packing Research Articles

Structural Basis for Effector Transmembrane Domain Recognition by Type VI Secretion System Chaperones

Type VI secretion systems facilitate the delivery of antibacterial effector proteins between neighbouring Gram-negative bacteria. A subset of these effectors harbour N-terminal transmembrane domains (TMDs) implicated in effector translocation across the target cell membrane. However, the abundance and distribution of these TMD-containing effectors has remained unknown. Here, we report the discovery of prePAAR, a conserved motif found in over 6,000 putative TMD-containing effectors. Based on their differing sizes and number of TMDs these effectors fall into two distinct classes that are unified by their requirement for a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain, which is required for effector interaction with the T6SS spike. Taken together, our findings define the mechanism of chaperone-assisted secretion of a widespread family of T6SS membrane protein effectors.

Hydraulics and mass transfer characteristics of novel helical liquid-bridge flow structured packings

Liquid bridges can be easily formed between two parallel slender plates. In a previous study, we constructed a similar semi-closed “channel” using a helical string and identified a liquid-bridge flow. Thus, the concept of helical liquid-bridge flow-structured packing was developed. Lab-scale fluid flow and distillation experiments were conducted to investigate the hydraulics and mass transfer characteristics, respectively. The test liquid mixtures could form a stable liquid-bridge flow in the channel of the tested helical string, which is the basic element of the novel packings. The structural parameters (width of the channel and diameter of the helical string) considerably affected the loading limit, liquid holdup, and residence time. The results indicate that these novel packings can offer a competitive separation efficiency but considerably lower pressure drop and better self-distribution, compared to commercial structured packings.

Hierarchical Self-Assembly of Nanowires on the Surface by Metallo-Supramolecular Truncated Cuboctahedra.

Parastichy, the spiral arrangement of plant organs, is an example of the long-range apparent order seen in biological systems. These ordered arrangements provide scientists with both an aesthetic challenge and a mathematical inspiration. Synthetic efforts to replicate the regularity of parastichy may allow for molecular-scale control over particle arrangement processes. Here we report the packing of a supramolecular truncated cuboctahedron (TCO) into double-helical (DH) nanowires on a graphite surface with a non-natural parastichy pattern ascribed to the symmetry of the TCOs and interactions between TCOs. Such a study is expected to advance our understanding of the design inputs needed to create complex, but precisely controlled, hierarchical materials. It is also one of the few reported helical packing structures based on Platonic or Archimedean solids since the discovery of the Boerdijk-Coxeter helix. As such, it may provide experimental support for studies of packing theory at the molecular level.

Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.

Insights into the Dynamics of the Human Zinc Transporter ZnT8 by MD Simulations.

ZnT8 is a human zinc(II) transporter expressed at the membrane of secretory granules where it contributes to insulin storage importing zinc ions from the cytosol. In the human population, the two most common ZnT8 variants carry an arginine (R325) or a tryptophan (W325) in position 325. The former variant has the most efficient kinetics in zinc transport and has been correlated to a higher risk of developing insulin resistance. On the contrary, the W325 variant is less active and protects against type-2-diabetes. Here, we used molecular dynamics (MD) simulations to investigate the main differences between the R325 and W325 variants in the interaction with zinc(II) ions. Our simulations suggested that the position of the metal ion within the transport site was not the same for the two variants, underlying a different rearrangement of the transmembrane (TM) helices in the channel. The W325 variant featured a peculiar zinc environment not detected in the experimental structures. With respect to conformational dynamics, we observed that the R325 variant was significantly more flexible than W325, with the main role played by the transmembrane domain (TMD) and the C-terminal domain (CTD). This dynamics affected the packing of the TM helices and thus the channel accessibility from the cytosol. The dimer interface that keeps the two TM channels in contact became looser in both variants upon zinc binding to the transport site, suggesting that this may be an important step toward the switch from the inward- to the outward-facing state of the protein.

Microphase separation in helix-coil block copolymer melts: computer simulation.

By means of molecular dynamics simulation, the process of the microphase separation in the melts of diblock helix-coil copolymers comprising a flexible and a helical block was studied. The resulting microstructures were examined, and the spatial distribution of the blocks and molecular packing were investigated. The phase diagram was built in terms of the fraction of the helical block and the incompatibility parameter of the blocks. The comparison of the diagrams for helix-coil and the classic coil-coil copolymer blends was carried out. It was shown that the total region where the ordering into distinctive microstructures takes place is similar for both diagrams. But for the helix-coil copolymers the area of the cylinders splits into the region of those with circular and elliptical cross-sections; the bicontinuous phase area is much wider; in the lamellar phases, the helical blocks were oriented precisely perpendicular to the lamellar interface, forming a cohesive interlocked structure of densely packed helices.

Unusual stoichiometry, band structure and band filling in conducting enantiopure radical cation salts of TM-BEDT-TTF showing helical packing of the donors

Enantiopure semiconducting crystalline radical cation salts of TM-BEDT-TTF with the Lindqvist type molybdenum based dianion, prepared by electrocrystallization, show unusual donor/anion ratio, charge distribution and helical packing of the donors.

Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones.

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

Different arrhythmia-associated calmodulin mutations have distinct effects on cardiac SK channel regulation.

Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.

Crystal structures of alpha and beta modifications of Mn as packing of tetrahedral helices extracted from a four-dimensional {3, 3, 5} polytope.

The crystal structures of both α- and β-Mn modifications have been presented as packing of tetrahedral helices extracted from four-dimensional {3, 3, 5} polytope construction. Presentation of the β-Mn structure as a primitive cubic arrangement formed by double tetrahedral helices around a central tetrahedral Coxeter-Boerdijk helix (tetrahelix) enables the inclusion in the structure description not only all atoms but also all tetrahedra; these tetrahedra are not accounted for in the preceding models for the β-Mn structure. The tetrahelix periodicity arising by minimal deformations of tetrahedra edges is equal to eight tetrahedra and coinciding with the lattice periods of both modifications. The linear substructure of α-Mn crystal consists of four tetrahelices which join to each other by edges around the common twofold axis. The α-Mn structure has been presented as primitive cubic arrangement constructed from such rods.

Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

Columnar Liquid Crystals from Star-Shaped Conjugated Mesogens as Nano-Reservoirs for Small Acceptors.

Shape-persistent conjugated mesogens with oligothiophene arms of different lengths have been synthesized. Such mesogens possess free intrinsic space between their conjugated arms. They form columnar liquid-crystalline phases, in which the void is filled by dense helical packing in the neat phase similar to an oligo(phenylene vinylene) derivative of equal size. The void can also be compensated by the inclusion of the small acceptor molecule 2,4,7-trinitrofluorenone. In solution, the acceptor interacts with the core as the largest π-surface, while in the solid material, it is incorporated between the arms and sandwiched by the star-shaped neighbours along the columnar assemblies. The TNF acceptors are not nanosegregated from the star-shaped donors, thus the liquid crystal structure converts to a nano-reservoir for TNF (endo-receptor). These host-guest arrangements are confirmed by comprehensive X-ray scattering experiments and solid-state NMR spectroscopy. This results in ordered columnar hexagonal phases at high temperatures, which change to helical columnar mesophases or to columnar soft crystals at room temperature.

On the role of the internal chain length distribution of amylopectins during retrogradation: Double helix lateral aggregation and slow digestibility

A structure-digestion model is proposed to explain the formation of α-amylase-slowly digestible structures during amylopectin retrogradation. Maize and potato (normal and waxy) and banana starch (normal and purified amylopectin through alcohol precipitation), were analyzed for amylose ratio and size (HPSEC) and amylopectin unit- and internal-chain length distribution (HPAEC). Banana amylopectin (BA), like waxy potato (WP), exhibited a larger number of B3-chains, fewer BS- and Bfp-chains and lower S:L and BS:BL ratios than maize, categorizing BA structurally as type-4. WP exhibited a significantly greater tendency to form double helices (DSC and 13C-NMR) than BA, which was attributed to its higher internal chain length (ICL) and fewer DP6−12-chains. However, retrograded BA was remarkably more resistant to digestion than WP. Lower number of phosphorylated B-chains, more S- and Bfp-chains and shorter ICL, were suggested to result in α-amylase-slowly digestible structures through further lateral packing of double helices (suggested by thermo-rheology) in type-4 amylopectins.

Toward Atomic-Scale Inorganic Double Helices via Carbon Nanotube Matrices—Induction of Chirality to Carbon Nanotubes

SnIP, an atomic-scale inorganic double helix compound is composed of a hexagonal rod packing of double helices in the bulk phase. A racemic mixture of P- and M-SnIP double helices is energetically ...

Self-Assembly of N-Terminal Aryl Amino Acids into Adaptive Single- and Double-Strand Helices.

Helical structures are important features of many important biomacromolecules such as double helices and single α-helices in DNA and protein, respectively, yet the self-organization of short oligopeptides (<3) or independent amino acids into artificial helical structures on the atomic level remains mysterious. Here we present the direct construction of artificial double and single helices from N-terminated aryl amino acids (ferrocene phenylalanine (Phe) conjugates) despite both Phe and Phe-Phe dipeptide self-aggregations adopting supramolecular β-sheet structures, which also demonstrated chirality evolution exposed to small molecular binders. In the solid state, the box-shaped building unit stacks into a double helix with enantiomer-resolved handedness driven orthogonally by H-bonds and the CH-π interaction. The entire double helix is noncovalently linked except for the hybridization regions. Asymmetric H-bonds between carboxylic acids and amides facilitates the one-dimensional helical packing of amino acid residues. The ditopic building unit adopts intramolecular H-bonds, facilitating single-strand helix formation. In aqueous self-assemblies, the superhelical structures were retained, which underwent chirality transfer and handedness inversion upon complexation orthogonally by H-bonds and charge-transfer interaction, showing adaptivity to environmental factors.

Effects of various oil extraction methods on the gelatinization and retrogradation properties of starches isolated from tigernut (Cyperus esculentus) tuber meals

Gelatinization and retrogradation characteristics of starches from tigernut (Cyperus esculentus) tuber before and after various oil extraction processes were studied in this investigation. The results indicated that starches isolated from tigernut tuber after the various oil extraction processes varied significantly in gelatinization and retrogradation properties. The starches isolated from the cakes of tigernut tuber after hot press extraction exhibited higher retrogradation tendency and relatively less shear-thinning than other starch samples. The results of FT-IR, XRD, and NMR analysis indicated that oil extraction had an unfavorable influence on starch retrogradation, which may be due to structural changes caused by oil extraction processes. In particular, oil extraction led to more efficient packing of double helices in the crystalline lamella of the starches during storage. Retrogradation of the starch gels also reduced the water holding capacities of the starches. The starch sample isolated from the cake after cold press extraction exhibited the highest water absorption capacity among the five samples for all storage times. This investigation provides valuable novel information for the industrial utilization of tigernut tuber starches isolated from meals and cakes after oil extraction.

Crystal Structure of a Proteolytic Fragment of the Sensor Histidine Kinase NarQ

Two-component signaling systems (TCSs) are a large and important class of sensory systems in bacteria, archaea, and some eukaryotes, yet their mechanism of action is still not fully understood from the structural point of view. Many TCS receptors are elongated flexible proteins with transmembrane (TM) regions, and are difficult to work with. Consequently, truncated fragments of the receptors are often used in structural studies. However, it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins. Recently, we crystallized a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase, NarQ, encompassing the sensor, TM, and HAMP domains. Here we report that a smaller proteolytic fragment consisting of the sensor and TM domains can also be crystallized using the in meso approach. The structure of the fragment is similar to the previously determined one, with minor differences in the vicinity of the truncation site. The results show that the crystallization of such sensor–TM fragments can be accomplished and can provide information on the packing of transmembrane helices, albeit limited, and that the proteolysis may or may not be a problem during crystallization.

Columnar Propeller‐Like 1,3,5‐Triphenylbenzenes: Probing the Effect of Chlorine on the Suzuki Cross‐Coupling and Liquid Crystalline Properties

Suzuki cross‐couplings either between chlorinated N‐methyliminodiacetic acid (MIDA)‐protected aryl boronates and 1,3,5‐tribromobenzene or between chlorinated aryl bromides and phenyltrisboronic species to star‐shaped 1,3,5‐triphenylbenzenes with different substitution patterns and chloro substituents at the outer phenyl rings were studied. The chlorinated precursors required for the respective reaction were synthesized and characterized. Depending on the used coupling reaction target triphenylbenzenes were isolated in yields between 42 % and 88 %. Their mesomorphic properties were influenced by the substitution pattern and number of peripheral chlorine atoms. Triphenylbenzene with 3,5‐alkoxy substitution and H in para‐position self‐assembled into either columnar hexagonal (Colh) mesophases or a soft crystal. While threefold chloro substitution in meta‐position of the outer phenyl rings led to stable room temperature Colho phases, triphenylbenzenes with threefold para‐chloro or 3,5‐dichloro substitution were non‐mesomorphic. Based on X‐ray diffraction data a helical packing model for the observed phases similar to that of related alkoxy‐substituted triphenylbenzenes was proposed.

Exploring Protein Fold Space.

The model of protein folding proposed by Ptitsyn and colleagues involves the accretion of secondary structures around a nucleus. As developed by Efimov, this model also provides a useful way to view the relationships among structures. Although somewhat eclipsed by later databases based on the pairwise comparison of structures, Efimov’s approach provides a guide for the more automatic comparison of proteins based on an encoding of their topology as a string. Being restricted to layers of secondary structures based on beta sheets, this too has limitations which are partly overcome by moving to a more generalised secondary structure lattice that can encompass both open and closed (barrel) sheets as well as helical packing of the type encoded by Murzin and Finkelstein on small polyhedra. Regular (crystalline) lattices, such as close-packed hexagonals, were found to be too limited so pseudo-latticses were investigated including those found in quasicrystals and the Bernal tetrahedron-based lattice that he used to represent liquid water. The Bernal lattice was considered best and used to generate model protein structures. These were much more numerous than those seen in Nature, posing the open question of why this might be.

Helix-helix interaction viewed in an angle frame indicates a role of the size of sidechains in packing

Three-dimensional packing is essential for protein folding. Homology alignment and de nova methods have been used to develop algorithms that would predict protein packing in proteins. Even though hydrophobic cores of proteins are formed through the packing of side chains of hydrophobic residues, the stereological arrangement of individual amino acids and in three dimensional hydrophobic cores is rather difficult to determine. In order to simplify the description of a protein core packing, in this manuscript, we have determined the relationship between angles, distances, and residue usage between two helices. Data from this analysis demonstrate that the contact residues determine the angle and the distance between two helical elements. This approach provides a means to predict the three-dimensional packing of helices in proteins and allows for an understanding of the biological interaction within proteins and among proteins based on surface contact residue parameters.