Helix Packing Research Articles

Clinical Phenotype in Individuals With Birk-Landau-Perez Syndrome Associated With Biallelic SLC30A9 Pathogenic Variants.

Birk-Landau-Perez syndrome is a genetic disorder caused by biallelic pathogenic variants in SLC30A9 presenting with a complex movement disorder, developmental regression, oculomotor abnormalities and renal impairment. So far it has been reported in only two families. We describe the clinical phenotype of eight further individuals from four unrelated families with SLC30A9-related disease. Following detailed clinical phenotyping, one family underwent research whole-genome sequencing (WGS), one research whole-exome sequencing (WES) and two diagnostic WGS. Variants of interest were assessed for pathogenicity using in silico prediction tools, homology modelling and where relevant, sequencing of cDNA for splicing impact. In two unrelated families of Pakistani origin (one consanguineous, one not), the same homozygous missense variant in SLC30A9 (c.1253G>T, p.Gly418Val) was identified. Family 1 included two affected brothers, and Family 2 one affected boy. In Family 3, also consanguineous, there were four affected siblings homozygous for the variant c.1049delCAG, pAla350del. The fourth family was non-consanguineous: the one affected individual was compound heterozygous for c.1083dup, p.Val362Cysfs*5 and c.1413A>G, p.Ser471=. Despite phenotypic variability between the four families, all affected patients manifested with a progressive hyperkinetic movement disorder, associated with oculomotor apraxia and ptosis. None had evidence of severe renal impairment. For the novel missense variant, the conformation of the loop domain and packing of transmembrane helices are likely to be disrupted based on structure modelling. Its presence in two unrelated Pakistani families suggests a possible founder variant. For the synonymous variant p.Ser471=, an impact on splicing was confirmed through cDNA analysis. Pathogenic variants in SLC30A9 cause a progressive autosomal recessive neurological syndrome associated with a complex hyperkinetic movement disorder. Our report highlights the expanding disease phenotype, which can present with a wider spectrum of severity than has previously been recognised.

Observing transmembrane conformational signaling with smFRET.

Transmembrane receptors transmit information about the local chemical environment across the membrane to regulate cellular signaling. The conformational changes responsible for signal transduction have been inaccessible due to the challenges associated with isolating and interrogating full-length receptors. As a result, studies have been limited to individual domains, where the transmembrane conformational dynamics are inherently missing. We overcame these limitations by embedding full-length receptors within membrane nanodiscs, a lipid bilayer encircled by a solubilizing belting protein, and using single-molecule Förster resonance energy transfer (FRET) to follow the transmembrane conformational changes through the receptor. First, in the aspartate chemoreceptor from E. coli we find a change in the cytoplasmic helical packing and dynamics upon ligand occupancy, which may be a general mechanism in coiled-coil signaling proteins. Second, in the mammalian epidermal growth factor receptor (EGFR), we identify a ligand- and lipid-dependent conformational change of the intracellular domain upon extracellular ligand binding, revealing a previously unknown transmembrane conformational coupling that, remarkably, is mediated by a single transmembrane alpha-helix.

Effects of Gly Residue and Cholesterol on the GXXXG-Mediated Parallel Association of Transmembrane Helices: A Single-Pair FRET Study.

Small residue-mediated interhelical packing is ubiquitous in helical membrane proteins: however, the lipid dependence of its stability remains unclear. We previously demonstrated that the introduction of a GXXXG sequence in the middle of de novo-designed (AALALAA)3 helices (AALALAA AGLALGA AALALAA) facilitated their dimerization, which was abolished by cholesterol. Here single-pair FRET measurements revealed that a longer GXXXGXXXG segment (AALALAA A GLALGA AAGALAA) promoted helix dimerization in POPC/cholesterol bilayers, but not without cholesterol. The predicted dimer structures and degrees of helix packing suggested that helix dimers with small (∼10°) and large (∼55°) crossing angles were only stabilized in POPC and POPC/cholesterol membranes, respectively. A steric hindrance in the dimer interface and the large flexibility of helices prevented the formation of stable dimers. Therefore, amino acid sequences and lipid compositions distinctively constrain stable dimer structures in membranes.

Topological examination of the bacteriophage lambda S holin by EPR spectroscopy

The S protein from bacteriophage lambda is a three-helix transmembrane protein produced by the prophage which accumulates in the host membrane during late gene expression. It is responsible for the first step in lysing the host cell at the end of the viral life cycle by multimerizing together to form large pores which permeabilize the host membrane to allow the escape of virions. Several previous studies have established a model for the assembly of holin into functional holes and the manner in which they pack together, but it is still not fully understood how the very rapid transition from monomer or dimer to multimeric pore occurs with such precise timing once the requisite threshold is reached. Here, site-directed spin labeling with a nitroxide label at introduced cysteine residues is used to corroborate existing topological data from a crosslinking study of the multimerized holin by EPR spectroscopy. CW-EPR spectral lineshape analysis and power saturation data are consistent with a three-helix topology with an unstructured C-terminal domain, as well as at least one interface on transmembrane domain 1 which is exposed to the lumen of the hole, and a highly constrained steric environment suggestive of a tight helical packing interface at transmembrane domain 2.

Distinguishment of Weak Interactions of Hydrogen Atoms Bound to Carbon Atoms: X-Ray Crystal Structural and Hirshfeld Surface Analyses of 2- Hydroxy-7-methoxy-3-(2,4,6-trimethylbenzoyl)naphthalene with the 2- Methoxylated Homologue

Abstract: X-ray crystal and Hirshfeld surface analyses of 2-hydroxy-7-methoxy-3-(2,4,6- trimethylbenzoyl)naphthalene and its 2-methoxylated homologue show quantitatively and visually distinct molecular contacts in crystals and minute differences in the weak intermolecular interactions. The title compound has a helical tubular packing, where molecules are piled in a two-folded head-to-tail fashion. The homologue has a tight zigzag molecular string lined up behind each other via nonclassical intermolecular hydrogen bonds between the carbonyl oxygen atom and the hydrogen atom of the naphthalene ring. The dnorm index obtained from the Hirshfeld surface analysis quantitatively demonstrates stronger molecular contacts in the homologue, an ethereal compound, than in the title compound, an alcohol, which is consistent with the higher melting temperature of the former than the latter. Stabilization through the significantly weak intermolecular nonclassical hydrogen bonding interactions in the homologue surpasses the stability imparted by the intramolecular C=O…H–O classical hydrogen bonds in the title compound. The classical hydrogen bond places the six-membered ring in the concave of the title molecule. The hydroxy group opposingly disturbs the molecular aggregation of the title compound, as demonstrated by the distorted H…H interactions covering the molecular surface, owing to the rigid molecular conformation. The position of effective interactions predominate over the strength of the classical/nonclassical hydrogen bonds in the two compounds.

A Uniquely Stable Trimeric Model of SARS-CoV-2 Spike Transmembrane Domain.

Understanding fusion mechanisms employed by SARS-CoV-2 spike protein entails realistic transmembrane domain (TMD) models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD only based on its primary structure. We performed amino acid sequence pattern matching and compared the molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and selected an appropriate template for homology modeling. We then iteratively built a model of spike TMD, adjusting “dynamic MHP portraits” and residue variability motifs. The stability of this model, with and without palmitoyl modifications downstream of the TMD, and several alternative configurations (including a recent NMR structure), was tested in all-atom molecular dynamics simulations in a POPC bilayer mimicking the viral envelope. Our model demonstrated unique stability under the conditions applied and conforms to known basic principles of TM helix packing. The original computational framework looks promising and could potentially be employed in the construction of 3D models of TM trimers for a wide range of membrane proteins.

Enzyme Responsive Rigid-Rod Aromatics Target "Undruggable" Phosphatases to Kill Cancer Cells in a Mimetic Bone Microenvironment.

Bone metastasis remains a challenge in cancer treatment. Here we show enzymatic responsive rigid-rod aromatics acting as the substrates of "undruggable" phosphatases to kill cancer cells in a mimetic bone microenvironment. By phosphorylation and conjugating nitrobenzoxadiazole (NBD) to hydroxybiphenylcarboxylate (BP), we obtained pBP-NBD (1P) as a substrate of both acid and alkaline phosphatases. 1P effectively kills both metastatic castration-resistant prostate cancer cells (mCRPCs) and osteoblast mimic cells in their coculture. 1P enters Saos2 almost instantly to target the endoplasmic reticulum (ER) of the cells. Co-culturing with Saos2 cells boosts the cellular uptake of 1P by mCRPCs. Cryo-EM reveals the nanotube structures of both 1P (2.4 Å resolution, pH 5.6) and 1 (2.2 Å resolution, pH 7.4). The helical packing of both nanotubes is identical, held together by strong pi-stacking interactions. Besides reporting the atomistic structure of nanotubes formed by the assembly of rigid-rod aromatics, this work expands the pool of molecules for designing EISA substrates that selectively target TME.

Helical Packing Columns for Preventing Foam Formation: Experimental and Numerical Investigations

AbstractUnwanted foaming and liquid bridging inside structured packings have a negative effect on the pressure drop during thermal separation processes. A novel helical design of a packing column for preventing foaming is presented. The effective interfacial area is calculated by numerical simulations. Different designs are analyzed by varying geometrical parameters such as helix pitch, channel opening angle, and number of channels. Different F‐factor values and liquid loads are evaluated. Packings with larger helix pitch exhibited lower pressure drop and reduced effective interfacial area. Smaller channel opening angles increased the pressure drop and promoted unstable flow conditions. The novel packings show lower effective interfacial area than existing structured packings, but no foaming was observed in a wide range of operating conditions.

Induction of a Columnar Liquid Crystal Phase at Low Temperature by Replacing Stearyl Groups with Oleyl Groups in a Discoid Molecule, and Efficient Chiral Amplification in the Liquid Crystal Phase

Abstract Replacement of stearyl groups with oleyl groups in a discoid molecule induced a rectangular columnar phase at low temperature. Although the molecule has no hydrogen bonding sites, the induced liquid crystal phase showed a highly efficient chiral amplification. The helical packing structure was postulated based on its X-ray diffraction profile and circular dichroism spectra.

The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding.

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following substrate binding. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. By contrast, an equilibrated structure does not show such classes. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties.

Simple Double Hetero[5]helicenes Realize Highly Efficient and Narrowband Circularly Polarized Organic Light-Emitting Diodes

Simple Double Hetero[5]helicenes Realize Highly Efficient and Narrowband Circularly Polarized Organic Light-Emitting Diodes

X⋅⋅⋅X Halogen Bond-Induced Supramolecular Helices.

The halogen bond is the attractive interaction between the electrophilic region of a halogen atom and the nucleophilic region of another molecular entity, emerging as a favorable manner to manipulate supramolecular chirality in self-assemblies. Engineering halogen bonded helical structures remains a challenge due to its sensitivity to solvent polarity and competitive forces like hydrogen bonds. Herein, we report a X⋅⋅⋅X (X=Cl, Br, I) type weak halogen bond that induces the formation and evolution of supramolecular helical structures both in solid and solution state. The π-conjugated phenylalanine derivatives with F, Cl, Br and I substitution self-assembled into 21 helical packing driven by hydrogen bond and halogen bond, respectively. The specific molecular geometries of π-conjugated amino acids gave rise to multiple noncovalent forces to stabilize the X⋅⋅⋅X halogen bond with small bond energies ranging from -0.69 to -1.49 kcal mol-1 . Halogen bond induced an opposite helicity compared to the fluorinated species, accompanied by the inversed circularly polarized luminescence.

X⋅⋅⋅X Halogen Bond‐Induced Supramolecular Helices

AbstractThe halogen bond is the attractive interaction between the electrophilic region of a halogen atom and the nucleophilic region of another molecular entity, emerging as a favorable manner to manipulate supramolecular chirality in self‐assemblies. Engineering halogen bonded helical structures remains a challenge due to its sensitivity to solvent polarity and competitive forces like hydrogen bonds. Herein, we report a X⋅⋅⋅X (X=Cl, Br, I) type weak halogen bond that induces the formation and evolution of supramolecular helical structures both in solid and solution state. The π‐conjugated phenylalanine derivatives with F, Cl, Br and I substitution self‐assembled into 21 helical packing driven by hydrogen bond and halogen bond, respectively. The specific molecular geometries of π‐conjugated amino acids gave rise to multiple noncovalent forces to stabilize the X⋅⋅⋅X halogen bond with small bond energies ranging from −0.69 to −1.49 kcal mol−1. Halogen bond induced an opposite helicity compared to the fluorinated species, accompanied by the inversed circularly polarized luminescence.

Chiral V‐shaped Pyrenes: Hexagonal Packing, Superhelix, and Amplified Chiroptical Performance

AbstractHere, we designed symmetric and dissymmetric chiral V‐shaped pyrenes by linking achiral pyrenes to trans‐1,2‐cyclohexane diamine scaffolds with varied spacers to investigate their circular dichroism (CD) and circularly polarized excimer emission (CPEE). In molecular solution, the symmetric V‐shaped molecules (P1, P2, P3) displayed spacer‐dependent CD and CPEE originating from the intramolecular excimers while the dissymmetric V‐shaped B was silent in CD and CPEE. Upon self‐assembly, the chiral V‐shaped conformation guided a helical hexagonal packing. Notably, P1 self‐assembled into delicate superhelices with optimum chiroptical activities and the largest gCD for pyrene derivatives to date. The dissymmetric B formed two distinct hexagonal aggregates as twists and rectangular nanotubes with greatly amplified CPEE. This work demonstrates unprecedented hexagonal superhelices from chiral V‐shaped scaffolds and provides a deep insight into the relationship between molecular conformation, supramolecular architectures, and their chiroptical performance.

Chiral V-shaped Pyrenes: Hexagonal Packing, Superhelix, and Amplified Chiroptical Performance.

Here, we designed symmetric and dissymmetric chiral V-shaped pyrenes by linking achiral pyrenes to trans-1,2-cyclohexane diamine scaffolds with varied spacers to investigate their circular dichroism (CD) and circularly polarized excimer emission (CPEE). In molecular solution, the symmetric V-shaped molecules (P1, P2, P3) displayed spacer-dependent CD and CPEE originating from the intramolecular excimers while the dissymmetric V-shaped B was silent in CD and CPEE. Upon self-assembly, the chiral V-shaped conformation guided a helical hexagonal packing. Notably, P1 self-assembled into delicate superhelices with optimum chiroptical activities and the largest gCD for pyrene derivatives to date. The dissymmetric B formed two distinct hexagonal aggregates as twists and rectangular nanotubes with greatly amplified CPEE. This work demonstrates unprecedented hexagonal superhelices from chiral V-shaped scaffolds and provides a deep insight into the relationship between molecular conformation, supramolecular architectures, and their chiroptical performance.

Computational design of a minimal “protein-like” conjugate for potent membrane poration

Biomimetic membrane nanopores have great potentials for bio- and nanotechnology applications. Here, a design strategy of an artificial protein by reasonably conjugating peptides and polymers for membrane poration was developed based on the α-helical bundle framework. A serial of peptide-polymer conjugates, composed of the α-helical peptide melittin (Mel) and clinically available polymer polyethylene glycol, were fabricated. Within them, the S-Mel, consisting of four helices arranged in a star-like bundle structure, demonstrated the most improved performance. Specifically, one single S-Mel molecule can build a stable membrane pore. Moreover, the obtained pores display stimuli-responsive properties in both size (including water flux) and structure, which is ascribed to the molecular architecture-regulated helical packing changes of S-Mel during membrane actions. These results promise S-Mel the ability to achieve ONOFF switching and gating effects for selective mass translocation across a cell membrane and help understand the structure-function relationship of proteins.

Loading Linear Arrays of CuII Inside Aromatic Amide Helices.

The very stable helices of 8‐amino‐2‐quinolinecarboxylic acid oligoamides are shown to uptake CuII ions in their cavity through deprotonation of their amide functions with minimal alteration of their shape, unlike most metallo‐organic structures which generally differ from their organic precursors. The outcome is the formation of intramolecular linear arrays of a defined number of CuII centers (up to sixteen in this study) at a 3 Å distance, forming a molecular mimic of a metal wire completely surrounded by an organic sheath. The helices pack in the solid state so that the arrays of CuII extend intermolecularly. Conductive‐AFM and cyclic voltammetry suggest that electrons are transported throughout the metal‐loaded helices in contrast with hole transport observed for analogous foldamers devoid of metal ions.

Loading Linear Arrays of CuII Inside Aromatic Amide Helices

AbstractThe very stable helices of 8‐amino‐2‐quinolinecarboxylic acid oligoamides are shown to uptake CuII ions in their cavity through deprotonation of their amide functions with minimal alteration of their shape, unlike most metallo‐organic structures which generally differ from their organic precursors. The outcome is the formation of intramolecular linear arrays of a defined number of CuII centers (up to sixteen in this study) at a 3 Å distance, forming a molecular mimic of a metal wire completely surrounded by an organic sheath. The helices pack in the solid state so that the arrays of CuII extend intermolecularly. Conductive‐AFM and cyclic voltammetry suggest that electrons are transported throughout the metal‐loaded helices in contrast with hole transport observed for analogous foldamers devoid of metal ions.

Structural Modeling and in planta Complementation Studies Link Mutated Residues of the Medicago truncatula Nitrate Transporter NPF1.7 to Functionality in Root Nodules.

Symbiotic nitrogen fixation is a complex and regulated process that takes place in root nodules of legumes and allows legumes to grow in soils that lack nitrogen. Nitrogen is mostly acquired from the soil as nitrate and its level in the soil affects nodulation and nitrogen fixation. The mechanism(s) by which legumes modulate nitrate uptake to regulate nodule symbiosis remain unclear. In Medicago truncatula, the MtNPF1.7 transporter has been shown to control nodulation, symbiosis, and root architecture. MtNPF1.7 belongs to the nitrate/peptide transporter family and is a symporter with nitrate transport driven by proton(s). In this study we combined in silico structural predictions with in planta complementation of the severely defective mtnip-1 mutant plants to understand the role of a series of distinct amino acids in the transporter’s function. Our results support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. Results reveal that Motif A, a motif conserved among major facilitator transport (MFS) proteins, is essential for function. We hypothesize that it participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. Our results also question the existence of a putative TMH4-TMH10 salt bridge. These results are discussed in the context of potential nutrient transport functions for MtNPF1.7. Our findings add to the knowledge of the mechanism of alternative conformational changes as well as symport transport in NPFs and enhance our knowledge of the mechanisms for nitrate signaling.

Functional and structural analyses of novel Smith-Kingsmore Syndrome-Associated MTOR variants reveal potential new mechanisms and predictors of pathogenicity.

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder characterized by macrocephaly/megalencephaly, developmental delay, intellectual disability, hypotonia, and seizures. It is caused by dominant missense mutations in MTOR. The pathogenicity of novel variants in MTOR in patients with neurodevelopmental disorders can be difficult to determine and the mechanism by which variants cause disease remains poorly understood. We report 7 patients with SKS with 4 novel MTOR variants and describe their phenotypes. We perform in vitro functional analyses to confirm MTOR activation and interrogate disease mechanisms. We complete structural analyses to understand the 3D properties of pathogenic variants. We examine the accuracy of relative accessible surface area, a quantitative measure of amino acid side-chain accessibility, as a predictor of MTOR variant pathogenicity. We describe novel clinical features of patients with SKS. We confirm MTOR Complex 1 activation and identify MTOR Complex 2 activation as a new potential mechanism of disease in SKS. We find that pathogenic MTOR variants disproportionately cluster in hotspots in the core of the protein, where they disrupt alpha helix packing due to the insertion of bulky amino acid side chains. We find that relative accessible surface area is significantly lower for SKS-associated variants compared to benign variants. We expand the phenotype of SKS and demonstrate that additional pathways of activation may contribute to disease. Incorporating 3D properties of MTOR variants may help in pathogenicity classification. We hope these findings may contribute to improving the precision of care and therapeutic development for individuals with SKS.