The presence of amyloid deposits consisting primarily of Amyloid-β (Aβ) fibril in the brain is a hallmark of Alzheimer's disease (AD). The morphologies of these fibrils are exquisitely sensitive to environmental conditions. Using molecular dynamics simulations combined with data from previously published solid-state NMR experiments, we propose the first atomically detailed structures of two asymmetric polymorphs of the Aβ9-40 peptide fibril. The first corresponds to synthetic fibrils grown under quiescent conditions and the second to fibrils derived from AD patients' brain-extracts. Our core structure in both fibril structures consists of a layered structure in which three cross-β subunits are arranged in six tightly stacked β-sheet layers with an antiparallel hydrophobic-hydrophobic and an antiparallel polar-polar interface. The synthetic and brain-derived structures differ primarily in the side-chain orientation of one β-strand. The presence of a large and continually exposed hydrophobic surface (buried in the symmetric agitated Aβ fibrils) may account for the higher toxicity of the asymmetric fibrils. Our model explains the effects of external perturbations on the fibril lateral architecture as well as the fibrillogenesis inhibiting action of amphiphilic molecules.
Amyloid diseases are characterized by the presence of amyloid fibrils on organs and tissue in the body. Alzheimer's disease, Parkinson's diseases and Type II Diabetes are all examples of amyloid diseases. Determining the structure of amyloid fibrils is critical for understanding the mechanism of fibril formation as well as for the design of inhibitor molecules that can prevent aggregation. In the case of the Alzheimer Amyloid-β (Aβ) peptide, the structure of fibrils grown under conditions of mechanical agitation has been elucidated from a combination of simulation and experiments. However, the structures of the asymmetric quiescent Aβ fibrils (grown under conditions akin to physiological conditions) and of Alzheimer's brain–derived fibrils are not known. In this paper, we propose the first atomically detailed structures of these two fibrils, using molecular dynamics simulations combined with data from previously published experiments. In additions, we suggest a unifying lateral growth mechanism that explains the increased toxicity of quiescent Aβ fibrils, the effects of external perturbations on fibril lateral architecture and the inhibition mechanism of the small molecule inhibitors on fibril formation.
At the core of amyloid fibrils is the cross-β spine, a long tape of β-sheets formed by the constituent proteins. Recent high-resolution x-ray studies show that the unit of this filamentous structure is a β-sheet bilayer with side chains within the bilayer forming a tightly interdigitating “steric zipper” interface. However, for a given peptide, different bilayer patterns are possible, and no quantitative explanation exists regarding which pattern is selected or under what condition there can be more than one pattern observed, exhibiting molecular polymorphism. We address the structural selection mechanism by performing molecular dynamics simulations to calculate the free energy of incorporating a peptide monomer into a β-sheet bilayer. We test filaments formed by several types of peptides including GNNQQNY, NNQQ, VEALYL, KLVFFAE and STVIIE, and find that the patterns with the lowest binding free energy correspond to available atomistic structures with high accuracy. Molecular polymorphism, as exhibited by NNQQ, is likely because there are more than one most stable structures whose binding free energies differ by less than the thermal energy. Detailed analysis of individual energy terms reveals that these short peptides are not strained nor do they lose much conformational entropy upon incorporating into a β-sheet bilayer. The selection of a bilayer pattern is determined mainly by the van der Waals and hydrophobic forces as a quantitative measure of shape complementarity among side chains between the β-sheets. The requirement for self-complementary steric zipper formation supports that amyloid fibrils form more easily among similar or same sequences, and it also makes parallel β-sheets generally preferred over anti-parallel ones. But the presence of charged side chains appears to kinetically drive anti-parallel β-sheets to form at early stages of assembly, after which the bilayer formation is likely driven by energetics.
Accumulation of amyloid fibrils is a salient feature of various protein misfolding diseases. Recent advances in precision experiments have begun to reveal their atomistic structures. Quantitative elucidation of how the observed structures are selected over other possible filament patterns would provide much insight into the formation and properties of amyloid fibrils. Using computer simulations and structural modeling, we demonstrate that the most stable filament pattern corresponds to the experimentally observed structure, and molecular polymorphism, selection of two or more patterns, is possible when there are more than one most stable structures. Ability to predict the structure allows for more detailed analysis, so that, for example, we can identify the most important residue for stabilizing the structure that could be therapeutically targeted. Our analysis will be useful for comparing different amyloid structures formed by the same protein or when delineating roles of different intermolecular forces in filament formation.
In this work, the graphene/α-SiO2(0001) interface is calculated using density functional theory. On the oxygen-terminated SiO2 surface, atomic structure reconstruction occurs at the graphene/SiO2 interface to eliminate the dangling bonds. The interface interaction is 77 meV/C atom, which indicates that van der Waals force dominates the interaction, but it is stronger than the force between the graphene layers in graphite. The distance between graphene and the SiO2 surface is 2.805 Å, which is smaller than the 3.4 Å interlayer distance of graphite. In addition, the SiO2 substrate induces p-type doping in graphene and opens a small gap of 0.13 eV at the Dirac point of graphene, which is desirable for electronic device applications.
Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e1 bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.
The cellular membrane functions as a regulating barrier between the intracellular and extracellular regions. For a molecule to reach the interior of the cell from the extracellular fluid, it must diffuse across the membrane, via either active or passive transport. The rigid structure of lipid bilayers, which are a key component of cellular membranes, prohibit simple diffusion of most particles, while vital nutrients are transported to the interior by specific mechanisms, such as ion channels and transport proteins. Although the cellular membrane provides the cell with protection against unwanted toxins that may be in the extracellular medium, some foreign particles can reach the interior of the cell, resulting in irregularities in cellular function. This behavior is particularly noted for permeants with compact molecular structure, suggesting that common nanoscale building blocks, such as fullerenes, may enter into the interior of a cell. To gauge the propensity for such particles to cross the membrane, we have computed the Gibbs free energy of transfer along the axis normal to the bilayer surface for two nanoscale building blocks, C60 and a hydrogen-terminated polyhedral oligomeric silsequioxane (H-POSS) monomer, in a hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer using molecular dynamics simulations and potential of mean force calculations. The studies show that C60 has a substantial energetic preference for the soft polymer region of the lipid bilayer system, below the water/bilayer interface, with a transition energy from bulk water of −19.8 kcal/mol. The transition of C60 from the bulk water to the center of the bilayer, while also energetically favorable, has to overcome a +5.9 kcal/mol energetic barrier in the hydrophobic lipid tail region. The H-POSS simulations indicate an energy minimum at the water-bilayer interface, with an energy of −10.9 kcal/mol; however, a local minimum of −2.7 kcal/mol is also observed in the hydrophobic dense-aliphatic region. The energy barrier seen in the hydrophobic core region of the C60 study is likely due to the significant penalty associated with inserting the relatively large particle into such a dense region. In contrast, while H-POSS is found to be subject to an energetic penalty upon insertion into the bilayer, the relatively small size of the H-POSS solute renders this penalty less significant. The energy barrier seen in the soft polymer region for the H-POSS monomer is primarily attributed to the lack of favorable solute-bilayer electrostatic interactions, which are present in the interfacial region, and fewer van der Waals interactions in the soft polymer region than the dense aliphatic region. These results indicate that C60 may partition into the organic phase of the DPPC/water system, given the favorable free energies in the soft polymer and dense aliphatic regions of the bilayer, while H-POSS is likely to partition near the water-bilayer interface, where the particle has low-energy electrostatic interactions with the polar head groups of the bilayer.
DPPC; fullerene C60; POSS; nanoparticle; molecular dynamics; potential of mean force; Gibbs free energy; solubility
Human apolipoprotein (apo) C-II is one of several lipid-binding proteins that self-assemble into fibrils and accumulate in disease-related amyloid deposits. A general characteristic of these amyloid deposits is the presence of lipids, known to modulate individual steps in amyloid fibril formation. ApoC-II fibril formation is activated by sub-micellar phospholipids but inhibited by micellar lipids. We examined the mechanism for the activation by sub-micellar lipids using the fluorescently-labelled, short-chain phospholipid, 1-dodecyl-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-2-hydroxy-glycero-3-phosphocholine (NBD-lyso-12-PC). Addition of submicellar NBD-lyso-12-PC increased the rate of fibril formation by apoC-II approximately two-fold. Stopped flow kinetic analysis using fluorescence detection and low, non-fibril forming concentrations of apoC-II indicated NBD-Lyso-12-PC binds rapidly, in the millisecond timescale, followed by the slower formation of discrete apoC-II tetramers. Sedimentation velocity analysis showed NBD-Lyso-12-PC binds to both apoC-II monomers and tetramers at approximately 5 sites per monomer with an average dissociation constant of approximately 10 μM. Mature apoC-II fibrils formed in the presence of NBD-Lyso-12-PC were devoid of lipid indicating a purely catalytic role for sub-micellar lipids in the activation of apoC-II fibril formation. These studies demonstrate the catalytic potential of small amphiphilic molecules to control protein folding and fibril assembly pathways.
The remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall.
The basic building unit of the title complex, CuI(DMSO)(PPh3), reproduced by a symmetry centre, leads to the rhomboid dimers in which the CuI atoms are in a tetrahedral geometry. The dimers are discrete molecules, but through weak intermolecular C—H⋯O interactions involving two adjacent DMSO ligands, a one-dimensional chain assembly is formed.
The centrosymmetric dinuclear title compound, [Cu2I2(C2H6OS)2(C18H15P)2], represents the first example of a CuI complex ligated by an O-bound dimethyl sulfoxide ligand. In the crystal, the two tetrahedrally coordinated CuI atoms are bridged by two μ2-iodido ligands in an almost symmetrical rhomboid geometry. The loose Cu⋯Cu contact of 2.9874 (8) Å is longer than the sum of the van der Waals radii of two Cu atoms (2.8 Å), excluding a significant cupriophilic interaction in the actual dimer. C—H⋯O and C—H⋯I hydrogen bonding interactions as well as C—H⋯π(aryl) interactions stabilize the three-dimensional supramolecular network.
crystal structure; dinuclear CuI complexes; iodide bridges; triphenylphosphane; DMSO
In the title complex, [Ni(C21H24N2O4)]·1.78H2O, the NiII ion has a slightly distorted planar geometry, coordinated by the two N and two O atoms of the tetradentate Schiff base ligand, with a mean deviation of 0.272 Å from the NiN2O2 plane. The N and O donor atoms are mutually cis. The dihedral angle between two benzene rings of the ligand is 38.86 (8)°. There are also three solvent water molecules, two of which lie across different crystallographic twofold rotation axes; one of these is partially occupied with a refined occupancy factor of 0.570 (7). The water molecules are linked together as tetramers in R
2(8) ring motifs, which also connect two neighbouring molecules of the complex through a network of O—H⋯O hydrogen bonds. The crystal structure is further stabilized by intermolecular C—H⋯O and C—H⋯π interactions, which link neighbouring molecules into extended chains along the b axis. Other interesting features of the crystal structure are the short intermolecular C⋯C [3.204 (3)–3.365 (3) Å] and the C⋯O [3.199 (2)–3.205 (2) Å] contacts which are shorter than the sum of the van der Waals radii of these atoms.
Misfolding and self-assembly of Amyloid-β (Aβ) peptides into amyloid fibrils is pathologically linked to the development of Alzheimer's disease. Polymorphic Aβ structures derived from monomers to intermediate oligomers, protofilaments, and mature fibrils have been often observed in solution. Some aggregates are on-pathway species to amyloid fibrils, while the others are off-pathway species that do not evolve into amyloid fibrils. Both on-pathway and off-pathway species could be biologically relevant species. But, the lack of atomic-level structural information for these Aβ species leads to the difficulty in the understanding of their biological roles in amyloid toxicity and amyloid formation.
Methods and Findings
Here, we model a series of molecular structures of Aβ globulomers assembled by monomer and dimer building blocks using our peptide-packing program and explicit-solvent molecular dynamics (MD) simulations. Structural and energetic analysis shows that although Aβ globulomers could adopt different energetically favorable but structurally heterogeneous conformations in a rugged energy landscape, they are still preferentially organized by dynamic dimeric subunits with a hydrophobic core formed by the C-terminal residues independence of initial peptide packing and organization. Such structural organizations offer high structural stability by maximizing peptide-peptide association and optimizing peptide-water solvation. Moreover, curved surface, compact size, and less populated β-structure in Aβ globulomers make them difficult to convert into other high-order Aβ aggregates and fibrils with dominant β-structure, suggesting that they are likely to be off-pathway species to amyloid fibrils. These Aβ globulomers are compatible with experimental data in overall size, subunit organization, and molecular weight from AFM images and H/D amide exchange NMR.
Our computationally modeled Aβ globulomers provide useful insights into structure, dynamics, and polymorphic nature of Aβ globulomers which are completely different from Aβ fibrils, suggesting that these globulomers are likely off-pathway species and explaining the independence of the aggregation kinetics between Aβ globulomers and fibrils.
We present herein characteristics of a conjugate in which dL5, a fluorogen-activating protein (FAP) and AEAEAKAK, an amphiphilic peptide are combined to form a solid-phase fluorescence-detection platform. The FAP dL5 is a covalently linked dimer of two identical light chain variable fragments which activates the fluorescence of the fluorogen malachite green (MG). The amphiphilic peptide of sequence AEAEAKAK is a building block of stimuli-responsive materials that undergoes sol-gel phase transition at high ionic strengths. We hypothesize that the novel bi-functional protein containing both the FAP and the amphiphile, termed dL5_EAK, co-assembles with the self-assembling peptide [AEAEAKAK]2 (EAK16-II) to form an insoluble membrane composite whereby the fluorescence enhancement function of the FAP domain remains intact. Denaturing polyacrylamide electrophoresis indicated that greater than 78% of dL5_EAK incorporates into the EAK16-II membrane. Conversely, less than 32% of dL5 without the EAK sequence associates with the insoluble fraction of EAK16-II in buffers. Membranes containing dL5_EAK and EAK16-II exhibited at least 4-fold higher fluorescence intensity compared to mixtures containing dL5 and EAK16-II. Scanning electron microscopy revealed the presence of particulates, presumably FAPs, scattering on the membrane fibrils. The evidence suggests a system of materials that can be developed into in situ-forming local sensors by immobilizing dL5 into coacervate, on which MG can be detected. It is envisioned that dL5 membranes can be established in diseased locales to monitor infiltration and migration of inflammatory cells marked with antibodies conjugated to MG.
The molecular structure of amyloid fibrils and the mechanism of their formation are of substantial medical and biological importance, but present an ongoing experimental and computational challenge. An early high-resolution view of amyloid-like structure was obtained on amyloid-like crystals of a small fragment of the yeast prion protein Sup35p: the peptide GNNQQNY. As GNNQQNY also forms amyloid-like fibrils under similar conditions, it has been theorized that the crystal's structural features are shared by the fibrils. Here we apply magic-angle-spinning (MAS) NMR to examine the structure and dynamics of these fibrils. Previously multiple NMR signals were observed for such samples, seemingly consistent with the presence of polymorphic fibrils. Here we demonstrate that peptides with these three distinct conformations instead assemble together into composite protofilaments. Electron-microscopy (EM) of the ribbon-like fibrils indicates that these protofilaments combine in differing ways to form striations of variable widths, presenting another level of structural complexity. Structural and dynamical NMR data reveal the presence of highly restricted side chain conformations involved in interfaces between differently structured peptides, likely comprising interdigitated steric zippers. We outline molecular interfaces that are consistent with the observed EM and NMR data. The rigid and uniform structure of the GNNQQNY crystals is found to contrast distinctly with the more complex structural and dynamic nature of these “composite” amyloid fibrils. These results provide insight into the fibril-crystal distinction and also indicate a necessary caution with respect to the extrapolation of crystal structures to the study of fibril structure and formation.
amyloid; solid state NMR; prion; fibrils; protofilament; fibrillization
We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the GLN side chain hydrogen bonding (HB) of a short, mainly polyGLN peptide of sequence D2Q10K2 (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that non-disaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) occurs in a β-sheet conformation, where the GLN side chains form HB to either the backbone or other GLN side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGLN disaggregation protocol of Wetzel et al (Methods Enzymol,
2006, 413, 34–74) to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.51-helix conformations where the GLN side chains form HB to water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon solution evaporation. The DQ10 PPII and 2.51-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded by NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril GLN side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.
Synthesis of a half metallic material on a substrate is highly desirable for diverse applications. Herein, we have investigated structural, adsorptive, and magnetic properties of metal free graphitic carbon nitride (g-C4N3) layer on hexagonal BN layer (h-BN) using the optB88-vdW van der Waals density functional theory. It is found that g-C4N3 layer can be adsorbed on BN layer due to the change of lattice constant of the hybridized system. The newly found lattice constant of g-C4N3 was 9.89 Å, which is approximately 2% lower and larger than to those of free standing BN and g-C4N3, respectively. Also, 2 × 2 surface reconstruction geometry predicted in free standing g-C4N3 layer disappears on the BN layer. Interestingly, we have found that metal free half metallic behavior in g-C4N3 can be preserved even on BN layer and the characters of spin polarized planar orbitals suggest that our theoretical prediction can be verified using normal incidence of K-edge X-ray magnetic circular dichroism (XMCD) measurement.
Based on ab initio calculations of both the ABC- and AB-stacked graphites, interlayer potentials (i.e., graphene-graphene interaction) are obtained as a function of the interlayer spacing using a modified Möbius inversion method, and are used to calculate basic physical properties of graphite. Excellent consistency is observed between the calculated and experimental phonon dispersions of AB-stacked graphite, showing the validity of the interlayer potentials. More importantly, layer-related properties for nonideal structures (e.g., the exfoliation energy, cleave energy, stacking fault energy, surface energy, etc.) can be easily predicted from the interlayer potentials, which promise to be extremely efficient and helpful in studying van der Waals structures.
Self-assembled, one-dimensional (1D) nanomaterials are amenable building blocks for bottom-up nanofabrication processes. A current shortcoming in the self-assembly of 1D nanomaterials in solution phase is the need for specific linkers or templates under very precise conditions to achieve a handful of systems. Here we report on the origin of a novel self-assembly of 1D dumbbells consisting of Au tipped PbS nanorods into stable chains in solution without any linkers or templates. A realistic multi-particle model suggests that the mesophase comprises 1D dumbbells arrayed in chains formed by anisotropic van der Waals type interactions. We demonstrate an alternative recognition mechanism for directing the assembly of the 1D dumbbells, based on effective interaction between the neighboring dumbbells consisting of Au tips with complementary crystallographic facets that guides the entire assembly in space.
The role of water in promoting the formation of protofilaments (the basic building blocks of amyloid fibrils) is investigated using fully atomic molecular dynamics simulations. Our model protofilament consists of two parallel β-sheets of Alzheimer Amyloid-β 16–22 peptides (Ac-K16-L17-V18-F19-F20-A21-E22-NH2). Each sheet presents a distinct hydrophobic and hydrophilic face and together self-assemble to a stable protofilament with a core consisting of purely hydrophobic residues (L17,F19,A21), with the two charged residues (K16, E22) pointing to the solvent. Our simulations reveal a subtle interplay between a water mediated assembly and one driven by favorable energetic interactions between specific residues forming the interior of the protofilament. A dewetting transition, in which water expulsion precedes hydrophobic collapse, is observed for some, but not all molecular dynamics trajectories. In the trajectories in which no dewetting is observed, water expulsion and hydrophobic collapse occur simultaneously, with protofilament assembly driven by direct interactions between the hydrophobic side chains of the peptides (particularly between F–F residues). For those same trajectories, a small increase in the temperature of the simulation (on the order of 20 K) or a modest reduction in the peptide–water van der Waals attraction (on the order of 10%) is sufficient to induce a dewetting transition, suggesting that the existence of a dewetting transition in simulation might be sensitive to the details of the force field parametrization.
Background: Protein oligomers may be toxic, but are unstable, difficult to study, and challenging therapeutic targets.
Results: Oligomers can be stabilized and used to generate and isolate antibodies with high selectivity for amyloid oligomers.
Conclusion: Oligomer stabilization is a powerful strategy to study and target amyloid oligomers.
Significance: We describe methods to generate oligomer-specific antibodies for molecular characterization, diagnosis and treatment of amyloid disorders.
The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.
Amyloid; Protease Inhibitor; Protein Aggregation; Protein Conformation; Protein Domains; Protein Misfolding; Cystatin C; Domain Swapping
Crystal structures of biosynthetic arginine decarboxylase (ADC, SpeA) from E. coli and C. jejuni are reported.
Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5′-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 Å resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 Å resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a β-sandwich domain, as well as two smaller helical domains. The TIM-barrel and β-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the β-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, π-stacking and van der Waals interactions.
biosynthetic arginine decarboxylases; SpeA; Campylobacter jejuni; Escherichia coli
Secreted phospholipase A2 (PLA2) plays a critical role in mobilizing aracidonic acid in phospholipids. We have previously reported that PLA2 is inhibited by B-type proanthocyanidins (PaC)s. To further understand the inhibitory activity of these compounds, we compared the inhibitory potency of B-type PaCs to that of A-type PaCs, and modeled them with PLA2 using in silico techniques. The B-type trimer and tetramer inhibited PLA2 (IC50 = 16 and 10 μM). The A-type compounds were less potent (18 – 35% inhibition at 50 μM). The active site of PLA2 lies in a hydrophobic tunnel. Modeling studies revealed that the B-type PaCs occupy this tunnel and are stabilized by a number of van der Waals interactions. The result is reduced substrate access to the active site. The A-type compounds can occupy this tunnel only by shifting the N-terminal loop outward. Our data provide a structural basis to screen additional PaCs for anti-PLA2 activity.
proanthocyanidin; phospholipase A2; inflammation; in silico modeling
Peptide amphiphile (PA) molecules that self-assemble in vivo into supramolecular nanofibers were used as a therapy in a mouse model of spinal cord injury (SCI). Because self-assembly of these molecules is triggered by the ionic strength of the in vivo environment, nanoscale structures can be created within the extracellular spaces of the spinal cord by simply injecting a liquid. The molecules are designed to form cylindrical nanofibers that display to cells in the spinal cord the laminin epitope IKVAV at nearly van der Waals density. IKVAV PA nanofibers are known to inhibit glial differentiation of cultured neural stem cells and to promote neurite outgrowth from cultured neurons.In this work, in vivo treatment with the PA after SCI reduced astrogliosis, reduced cell death, and increased the number of oligodendroglia at the site of injury. Furthermore, the nanofibers promoted regeneration of both descending motor fibers and ascending sensory fibers through the lesion site.Treatment with the PA also resulted in significant behavioral improvement. These observations demonstrate that it is possible to inhibit glial scar formation and to facilitate regeneration after SCI using bioactive three-dimensional nanostructures displaying high densities of neuroactive epitopes on their surfaces.
spinal cord injury; nanotechnology; gliosis; regeneration; extracellular matrix; functional recovery
Recent experiments have shown that the congener, Aβ1–40[D23-K28], in which the side chains of charged residues Asp23 and Lys28 are linked by a lactam bridge, forms amyloid fibrils that are structurally similar to the wild type (WT) Aβ peptide, but at a rate that is nearly thousand times faster. We used all atom molecular dynamics in explicit water, and two force fields, of the WT dimer, a monomer with the lactam bridge (Aβ10–35-lactam[D23-K28]), the monomer and dimers with harmonically constrained D23-K28 salt bridge (Aβ10–35[D23-K28]), to understand the origin of the enhanced fibril rate formation. The simulations show that the assembly-competent fibril like monomer (N*) structure, that is present among the conformations sampled by the isolated monomer, with strand conformations in the residues spanning the N and C termini and a bend involving residues D23VGSNKG29, are populated to a much greater extent in Aβ10–35[D23-K28] and Aβ10–35-lactam[D23-K28] than in the WT, which has negligible probability of forming N*. The salt bridge in N* of Aβ10–35[D23-K28], whose topology is similar to that found in the fibril, is hydrated. The reduction in the free energy barrier to fibril formation in Aβ10–35[D23-K28] and in Aβ10–35-lactam[D23-K28], compared to the WT, arises largely due to entropic restriction that enables the bend formation. A decrease in the entropy of the unfolded state and the lesser penalty for conformational rearrangement including the formation of the salt bridge in Aβ peptides with D23-K28 constraint results in a reduction in the kinetic barrier in the Aβ1–40-lactam[D23-K28] congener compared to the WT. The decrease in the barrier, that is related to the free energy cost of forming a bend, is estimated to be in the range (4–7)kBT. Although a number of factors determine the growth of fibrils, the decrease in the free energy barrier, relative to the WT, to N* formation is a major factor in the rate enhancement in the fibril formation of Aβ1–40[D23-K28] congener. Qualitatively similar results were obtained using simulations of Aβ9–40 peptides, and various constructs related to the Aβ10–35 systems that were probed using OPLS and CHARMM force fields. We hypothesize that mutations or other constraints that preferentially enhance the population of N* species would speed up aggregation rates. Conversely, ligands that lock it in the fibril-like N* structure would prevent amyloid formation.
β-2-microglobulin (β2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes β2m to organize into native-like dimers and tetramers that precede amyloid formation. Cu(II) is then released from higher order oligomers before mature Cu(II)-free amyloid fibrils are formed. While some of the Cu(II)-induced structural changes that enable β2m self assembly are starting to be revealed, the details of how the Cu(II) binding site evolves from the monomer to the dimers and tetramers are not known. Here, we report results from three mass spectrometry (MS) based methods that provide insight into the changing Cu-β2m interactions. We find that monomeric β2m binds Cu(II) via the N-terminal amine, the amide of Gln2, His31, and Asp59. In the dimer and tetramer, Asp59 is no longer bound to Cu(II), but the other residues still comprise a well-defined albeit weaker binding site that is better able to release Cu(II). Consistent with this is the observation that a fraction of the tetrameric species no longer binds Cu(II) at this weakened binding site, which agrees with a previous report that suggested the tetramer as the first Cu(II)-free oligomer. Our results also provide some insight into structural changes caused by Cu(II) binding that facilitate oligomer formation. Specifically, binding by Asp59 in the monomer requires significant movement of this residue, and we propose that this repositioning is important for establishing a pair of dimer-stabilizing salt bridges between this residue and Lys19. We also find evidence that Cu(II) binding in the N-terminal region of the monomer repels Arg3, which likely allows this residue to form a pair of dimer-stabilizing salt bridges with Glu16. Overall, our measurements suggest that the previously proposed conformational switch caused by Cu(II) binding includes not only a cis-trans isomerization at Pro32 but also the repositioning of residues that are critical for the formation of new electrostatic interactions.
Hydroxyproline-rich glycoproteins (HRGP) comprise a super-family of extracellular structural glycoproteins whose precise roles in plant cell wall assembly and functioning remain to be elucidated. However, their extended structure and repetitive block co-polymer character of HRGPs may mediate their self-assembly as wall scaffolds by like-with-like alignment of their hydrophobic peptide and hydrophilic glycopeptide modules. Intermolecular crosslinking further stabilizes the scaffold. Thus the design of HRGP-based scaffolds may have practical applications in bionanotechnology and medicine. As a first step, we have used single-molecule or single-aggregate atomic force microscopy (AFM) to visualize the structure of YK20, an amphiphilic HRGP comprised entirely of 20 tandem repeats of: Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr-Tyr-Tyr-Lys. YK20 formed tightly aggregated coils at low ionic strength, but networks of entangled chains with a porosity of ~0.5–3 μm at higher ionic strength. As a second step we have begun to design HRGP-carbon nanotube composites. Single-walled carbon nanotubes (SWNTs) can be considered as seamless cylinders rolled up from graphene sheets. These unique all-carbon structures have extraordinary aromatic and hydrophobic properties and form aggregated bundles due to strong inter-tube van der Waals interactions. Sonicating aggregated SWNT bundles with aqueous YK20 solubilized them presumably by interaction with the repetitive, hydrophobic, Tyr-rich peptide modules of YK20 with retention of the extended polyproline-II character. This may allow YK20 to form extended structures that could potentially be used as scaffolds for site-directed assembly of nanomaterials.
Hydroxyproline-rich glycoprotein; Carbon nanotube; Nano assembly
To assess whether there are universal rules that govern amino
acid–base recognition, we investigate hydrogen bonds, van
der Waals contacts and water-mediated bonds in 129 protein–DNA
complex structures. DNA–backbone interactions are the most numerous,
providing stability rather than specificity. For base interactions,
there are significant base–amino acid type correlations,
which can be rationalised by considering the stereochemistry of
protein side chains and the base edges exposed in the DNA structure.
Nearly two-thirds of the direct read-out of DNA sequences involves
complex networks of hydrogen bonds, which enhance specificity. Two-thirds
of all protein–DNA interactions comprise van der Waals contacts,
compared to about one-sixth each of hydrogen and water-mediated
bonds. This highlights the central importance of these contacts
for complex formation, which have previously been relegated to a secondary
role. Although common, water-mediated bonds are usually non-specific,
acting as space-fillers at the protein–DNA interface. In
conclusion, the majority of amino acid–base interactions observed
follow general principles that apply across all protein–DNA
complexes, although there are individual exceptions. Therefore,
we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’.
An interactive Web-based atlas of side chain–base contacts provides
access to the collected data, including analyses and visualisation
of the three-dimensional geometry of the interactions.