Higher-order Assemblies Research Articles

Higher-order SPOP assembly reveals a basis for cancer mutant dysregulation

The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP mutations have been attributed to the development of many types of cancers, including prostate and endometrial cancers. Prostate cancer mutations localize in the substrate-binding site of the substrate recognition (MATH) domain and reduce or prevent binding. However, most endometrial cancer mutations are dispersed in seemingly inconspicuous solvent-exposed regions of SPOP, offering no clear basis for their cancer-causing and peculiar gain-of-function properties. Herein, we present the first structure of SPOP in its oligomeric form, uncovering several new interfaces important for SPOP self-assembly and normal function. Given that many previously unaccounted-for cancer mutations are localized in these newly identified interfaces, we uncover molecular mechanisms underlying dysregulation of SPOP function, with effects ranging from gross structural changes to enhanced self-association, and heightened stability and activity.

Lysine Acetylation Changes the Mechanism of Aβ25-35 Peptide Binding and Dimerization in the DMPC Bilayer.

The impact of Lys28 acetylation on Alzheimer's Aβ peptide binding to the lipid bilayer has not been previously studied, either experimentally or computationally. To probe this common post-translational modification, we performed all-atom replica exchange molecular dynamics simulations targeting binding and aggregation of acetylated acAβ25-35 peptide within the DMPC bilayer. Using the unmodified Aβ25-35 studied previously as a reference, our results can be summarized as follows. First, Lys28 acetylation strengthens the Aβ25-35 hydrophobic moment and consequently promotes the helical structure across the peptide extending it into the N-terminus. Second, because Lys28 acetylation disrupts electrostatic contact between Lys28 and lipid phosphate groups, it reduces the binding affinity of acAβ25-35 peptides to the DMPC bilayer. Accordingly, although acetylation preserves the bimodal binding featuring a preferred inserted state and a less probable surface bound state, it decreases the stability of the former. Third, acetylation promotes acAβ25-35 aggregation and eliminates monomers as thermodynamically viable species. More importantly, acAβ25-35 retains as the most thermodynamically stable the inserted dimer with unique head-to-tail helical aggregation interface. However, due to enhanced helix structure, this dimer state becomes less stable and is less likely to propagate into higher order aggregates. Thus, acetylation is predicted to facilitate the formation of low-molecular-weight oligomers. Other post-translational modifications, including phosphorylation and oxidation, reduce helical propensity and have divergent impact on aggregation. Consequently, acetylation, when considered in its totality, has distinct consequences on Aβ25-35 binding and aggregation in the lipid bilayer.

Ocular Amyloid, Condensates, and Aggregates - Higher-Order Protein Assemblies Participate in Both Retinal Degeneration and Function.

The formation of higher-order protein assemblies (commonly called protein aggregates) has long been associated with disease states, particularly in neurodegenerative disorders. Within the eye, protein aggregation has also been implicated in various retinal degenerative diseases ranging from retinitis pigmentosa (RP) to Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) to age-related macular degeneration (AMD). Yet, many essential cellular processes including transcription, translation, and the formation of non-membrane bound organelles require the formation of functional, non-pathologic protein aggregates to maintain cellular homeostasis. Thus, functional protein aggregates, also called condensates, likely play essential roles in maintaining normal retina function. However, currently, there is a critical gap in our knowledge: What proteins form higher-order assemblies under normal conditions within the retina and what function do these structures serve? Herein, we present data suggesting that protein aggregation is identifiable in multiple retinal layers of normal, healthy murine retina, and briefly discuss the potential contributions of aggregated proteins to normal retinal function, with a focus on the photoreceptor inner and outer segment.

The effect of polymer end-group on the formation of styrene - maleic acid lipid particles (SMALPs).

A series of block copolymers comprising styrene and maleic acid (SMA) has been prepared using RAFT polymerisation. RAFT often results in a large hydrophobic alkylthiocarbonylthio end group and this work examines its effect on the solution behaviour of the copolymers. SMA variants with, and without, this end group were synthesised and their behaviour compared with a commercially-available random copolymer of similar molecular weight. Dynamic light scattering and surface tension measurements found the RAFT-copolymers preferentially self-assembled into higher-order aggregates in aqueous solution. Small angle neutron scattering using deuterated styrene varients add support to the accepted model that these agreggates comprise a solvent-protected styrenic core with an acid-rich shell. Replacing the hydrophobic RAFT end group with a more hydrophilic nitrile caused differences in the resulting surface activity, attributed to the ability of the adjoining styrene homoblock to drive aggregation. Each of the copolymers formed SMALP nanodiscs with DMPC lipids, which were found to encapsulate a model membrane protein, gramicidin. However, end group variation affected solubilisition of DPPC, a lipid with a higher phase transition temperature. When using RAFT-copolymers terminated with a hydrophobic group, swelling of the bilayer and greater penetration of the homoblock into the nanodisc core occurred with increasing homoblock length. Conversely, commercial and nitrile-terminated RAFT-copolymers produced nanodisc sizes that stayed constant, instead indicating interaction at the edge of the lipid patch. The results highlight how even minor changes to the copolymer can modify the amphiphilic balance between regions, knowledge useful towards optimising copolymer structure to enhance and control nanodisc formation.

Comparative study of the self-assembly behaviour of 3-chloro-4-hydroxy-phenylazo dyes.

The complexity of intermolecular interactions and the difficulty to predict assembly behaviour solely based on chemical constitution was demonstrated by studying the self-assembly of three one-fold negatively charged 3-chloro-4-hydroxy-phenylazo dyes (Yellow, Blue and Red). Dye self-assembly was investigated using UV/vis- and NMR-spectroscopy, light- and small-angle neutron scattering. Significant differences between the three dyes were observed. While Yellow does not self-assemble, Red assembles into higher-order aggregates and Blue forms well-defined H-aggregate dimers with a dimerization constant of KD = (728 ± 8) L mol-1. Differences between dyes were suggested to emerge from variations in the propensity to form π-π-interactions due to electrostatic repulsion, sterical constraints and hydrogen-bonding interactions.

Tuning a coiled-coil hydrogel via computational design of supramolecular fiber assembly

Higher-order supramolecular assembly of a coiled-coil protein can be modulated by iterative tuning of its electrostatic potential.

High order assembly of multiple protein cages with homogeneous sizes and shapes via limited cage surface engineering.

Protein cages are attractive building blocks to build high order materials such as 3D cage lattices, which offer accurately ordered bio-templates. However, controlling the size or valency of these cage-to-cage assemblies is extremely difficult due to highly multivalent and symmetric cage structures. Here, various high order cage assemblies with homogeneous sizes and geometries are constructed by developing an anisotropic ferritin cage with limitedly exposed binding modules, leucine zipper. The anisotropic ferritin is produced as expressed in cells without the need of complex in vitro cage fabrication by careful subunit manipulation. Ferritin cages with limitedly exposed zippers are assembled around a core ferritin with fully exposed opposing zippers, generating homogeneous high order structures, whereas two fully exposed ferritins are assembled into heterogeneous cage aggregates. Diverse fully exposed core cages are prepared by varying the zipper-ferritin fusion geometries and even by using larger cage structures. With these core cages and the anisotropic ferritin, a range of high order cage assemblies with diverse ferritin valencies (3 to over 12) and sizes (over 40 nm) are created. Cell surface binding and internalization of cage structures are greatly varied by assembly sizes, where high order ferritins are clearly more effective than monomeric ferritin.

DNA origami traps for large viruses

Virus-enveloping macromolecular shells or tilings can prevent viruses from entering cells. Here, we describe the design and assembly of a cone-shaped DNA origami higher-order assembly that can engulf and tile the surface of pleomorphic virus samples larger than 100 nm. We determine the structures of subunits and of complete cone assemblies using cryoelectron microscopy (cryo-EM) and establish stabilization treatments to enable usage in in vivo conditions. We use the cones exemplarily to engulf influenza A virus particles and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), chikungunya, and Zika virus-like particles. Depending on the relative dimensions of cone to virus particles, multiple virus particles may be trapped per single cone, and multiple cones can also tile and adapt to the surface of aspherical virus particles. The cone assemblies form with high yields, require little purification, and are amenable for mass production, which is a key requirement for future real-world uses including as a potential antiviral agent.

Fabrication of Protein Macromolecular Frameworks (PMFs) and Their Application in Catalytic Materials.

The construction of three-dimensional (3D) array materials from nanoscale building blocks has drawn significant interest because of their potential to exhibit collective properties and functions arising from the interactions between individual building blocks. Protein cages such as virus-like particles (VLPs) have distinct advantages as building blocks for higher-order assemblies because they are extremely homogeneous in size and can be engineered with new functionalities by chemical and/or genetic modification. In this chapter, we describe a protocol for constructing a new class of protein-based superlattices, called protein macromolecular frameworks (PMFs). We also describe an exemplary method to evaluate the catalytic activity of enzyme-enclosed PMFs, which exhibit enhanced catalytic activity due to the preferential partitioning of charged substrates into the PMF.

Mitochondrial supercomplex assembly regulates metabolic features and glutamine dependency in mammalian cells.

Rationale: Mitochondria generate ATP via the oxidative phosphorylation system, which mainly comprises five respiratory complexes found in the inner mitochondrial membrane. A high-order assembly of respiratory complexes is called a supercomplex. COX7A2L is a supercomplex assembly factor that has been well-investigated for studying supercomplex function and assembly. To date, the effects of mitochondrial supercomplexes on cell metabolism have not been elucidated. Methods: We depleted COX7A2L or Cox7a2l in human and mouse cells to generate cell models lacking mitochondrial supercomplexes as well as in DBA/2J mice as animal models. We tested the effect of impaired supercomplex assembly on cell proliferation with different nutrient supply. We profiled the metabolic features in COX7A2L-/- cells and Cox7a2l-/- mice via the combined use of targeted and untargeted metabolic profiling and metabolic flux analysis. We further tested the role of mitochondrial supercomplexes in pancreatic ductal adenocarcinoma (PDAC) through PDAC cell lines and a nude mouse model. Results: Impairing mitochondrial supercomplex assembly by depleting COX7A2L in human cells reprogrammed metabolic pathways toward anabolism and increased glutamine metabolism, cell proliferation and antioxidative defense. Similarly, knockout of Cox7a2l in DBA/2J mice promoted the use of proteins/amino acids as oxidative carbon sources. Mechanistically, impaired supercomplex assembly increased electron flux from CII to CIII/CIV and promoted CII-dependent respiration in COX7A2L-/- cells which further upregulated glutaminolysis and glutamine oxidation to accelerate the reactions of the tricarboxylic acid cycle. Moreover, the proliferation of PDAC cells lacking COX7A2L was inhibited by glutamine deprivation. Conclusion: Our results reveal the regulatory role of mitochondrial supercomplexes in glutaminolysis which may fine-tune the fate of cells with different nutrient availability.

Plant-specific features of respiratory supercomplex I + III2 from Vigna radiata

The last steps of cellular respiration—an essential metabolic process in plants—are carried out by mitochondrial oxidative phosphorylation. This process involves a chain of multi-subunit membrane protein complexes (complexes I–V) that form higher-order assemblies called supercomplexes. Although supercomplexes are the most physiologically relevant form of the oxidative phosphorylation complexes, their functions and structures remain mostly unknown. Here we present the cryogenic electron microscopy structure of the supercomplex I + III2 from Vigna radiata (mung bean). The structure contains the full subunit complement of complex I, including a newly assigned, plant-specific subunit. It also shows differences in the mitochondrial processing peptidase domain of complex III2 relative to a previously determined supercomplex with complex IV. The supercomplex interface, while reminiscent of that in other organisms, is plant specific, with a major interface involving complex III2’s mitochondrial processing peptidase domain and no participation of complex I’s bridge domain. The complex I structure suggests that the bridge domain sets the angle between the enzyme’s two arms, limiting large-scale conformational changes. Moreover, complex I’s catalytic loops and its response in active-to-deactive assays suggest that, in V. radiata, the resting complex adopts a non-canonical state and can sample deactive- or open-like conformations even in the presence of substrate. This study widens our understanding of the possible conformations and behaviour of complex I and supercomplex I + III2. Further studies of complex I and its supercomplexes in diverse organisms are needed to determine the universal and clade-specific mechanisms of respiration.

Protein interactions: anything new?

How do proteins interact in the cellular environment? Which interactions stabilize liquid-liquid phase separated condensates? Are the concepts, which have been developed for specific protein complexes also applicable to higher-order assemblies? Recent discoveries prompt for a universal framework for protein interactions, which can be applied across the scales of protein communities. Here, we discuss how our views on protein interactions have evolved from rigid structures to conformational ensembles of proteins and discuss the open problems, in particular related to biomolecular condensates. Protein interactions have evolved to follow changes in the cellular environment, which manifests in multiple modes of interactions between the same partners. Such cellular context-dependence requires multiplicity of binding modes (MBM) by sampling multiple minima of the interaction energy landscape. We demonstrate that the energy landscape framework of protein folding can be applied to explain this phenomenon, opening a perspective toward a physics-based, universal model for cellular protein behaviors.

Ubiquitin-assisted phase separation of dishevelled-2 promotes Wnt signalling.

Dishvelled-2 (Dvl2) is an essential component of Wnt pathway, which controls several cell fate decisions during development, such as proliferation, survival and differentiation. Dvl2 forms higher-order protein assemblies in the cell that are critical for relaying the signal from upstream Wnt ligand-frizzled receptor binding to downstream effector β-catenin activation. However, the precise molecular nature and contribution of Dvl2 protein assemblies during Wnt signalling is unknown. Here, we show that Dvl2 forms protein condensates driven by liquid-liquid phase separation. An intrinsically disordered region (IDR) at the N-terminus is essential for Dvl2 phase separation. Importantly, we identified the HECT-E3 ligase WWP2 as an essential driver of Dvl2 phase separation in vitro and in cells. We demonstrated that ubiquitylation of Dvl2 through K63 linkage by WWP2 is required for formation of Dvl2 condensates. Phase-separated Dvl2 activates Wnt signaling by sequestering the components of destruction complex and thus relieving β-catenin. Together, our results reveal a ubiquitylation-dependent liquid-liquid phase separation as a new process through which Dvl2 forms condensates, which is necessary for transduction of Wnt signalling. This article has an associated First Person interview with the first author of the paper.

A novel non-linear neuron model based on multiplicative aggregation in quaternionic domain

The learning algorithm for a three-layered neural structure with novel non-linear quaternionic-valued multiplicative (QVM) neurons is proposed in this paper. The computing capability of non-linear aggregation in the cell body of biological neurons inspired the development of a non-linear neuron model. However, unlike linear neuron models, most non-linear neuron models are built on higher order aggregation, which is more mathematically complex and difficult to train. As a result, building non-linear neuron models with a simple structure is a difficult and time-consuming endeavor in the neurocomputing field. The concept of a QVM neuron model was influenced by the non-linear neuron model, which has a simple structure and the great computational ability. The suggested neuron’s linearity is determined by the weight and bias associated with each quaternionic-valued input. Non-commutative multiplication of all linearly connected quaternionic input-weight terms accommodates the non-linearity. To train three-layered networks with QVM neurons, the standard quaternionic-gradient-based backpropagation (QBP) algorithm is utilized. The computational and generalization capabilities of the QVM neuron are assessed through training and testing in the quaternionic domain utilizing benchmark problems, such as 3D and 4D chaotic time-series predictions, 3D geometrical transformations, and 3D face recognition. The training and testing outcomes are compared to conventional and root-power mean (RPM) neurons in quaternionic domain using training–testing MSEs, network topology (parameters), variance, and AIC as statistical measures. According to these findings, networks with QVM neurons have greater computational and generalization capabilities than networks with conventional and RPM neurons in quaternionic domain.

Proline C-H Bonds as Loci for Proline Assembly via C-H/O Interactions.

Proline residues within proteins lack a traditional hydrogen bond donor. However, the hydrogens of the proline ring are all sterically accessible, with polarized C-H bonds at Hα and Hδ that exhibit greater partial positive character and can be utilized as alternative sites for molecular recognition. C-H/O interactions, between proline C-H bonds and oxygen lone pairs, have been previously identified as modes of recognition within protein structures and for higher-order assembly of protein structures. In order to better understand intermolecular recognition of proline residues, a series of proline derivatives was synthesized, including 4R-hydroxyproline nitrobenzoate methyl ester, acylated on the proline nitrogen with bromoacetyl and glycolyl groups, and Boc-4S-(4-iodophenyl)hydroxyproline methyl amide. All three derivatives exhibited multiple close intermolecular C-H/O interactions in the crystallographic state, with H⋅⋅⋅O distances as close as 2.3 Å. These observed distances are well below the 2.72 Å sum of the van der Waals radii of H and O, and suggest that these interactions are particularly favorable. In order to generalize these results, we further analyzed the role of C-H/O interactions in all previously crystallized derivatives of these amino acids, and found that all 26 structures exhibited close intermolecular C-H/O interactions. Finally, we analyzed all proline residues in the Cambridge Structural Database of small-molecule crystal structures. We found that the majority of these structures exhibited intermolecular C-H/O interactions at proline C-H bonds, suggesting that C-H/O interactions are an inherent and important mode for recognition of and higher-order assembly at proline residues. Due to steric accessibility and multiple polarized C-H bonds, proline residues are uniquely positioned as sites for binding and recognition via C-H/O interactions.

Constructing Divisia Monetary Aggregates for Nigeria

This study computes Divisia monetary aggregates DM1, DM2, and DM3 for Nigeria using the Tornqvist-Theil quantity index for the period 2007M12 to 2020M12 and evaluates the performance of the higher-order aggregate (DM3) with a corresponding higher aggregate of the simple sum broad money supply (M3). Applying the indicators to money demand function the results showed that DM3 performed better than the matching simple sum measure M3 due to its faster convergence rate. This is because the Divisia aggregates contain additional information in the form of user-costs and spending shares that are not considered in the compilation of the traditional measure, information that may be important for the proper execution of monetary policy. It is therefore, recommended that the Divisia broad money index be used as complement to the simple sum broad money supply for policy decisions.

The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy.

Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.

Protein cargo encapsulation by virus-like particles: Strategies and applications.

Viruses and the recombinant protein cages assembled from their structural proteins, known as virus-like particles (VLPs), have gained wide interest as tools in biotechnology and nanotechnology. Detailed structural information and their amenability to genetic and chemical modification make them attractive systems for further engineering. This review describes the range of non-enveloped viruses that have been co-opted for heterologous protein cargo encapsulation and the strategies that have been developed to drive encapsulation. Spherical capsids of a range of sizes have been used as platforms for protein cargo encapsulation. Various approaches, based on native and non-native interactions between the cargo proteins and inner surface of VLP capsids, have been devised to drive encapsulation. Here, we outline the evolution of these approaches, discussing their benefits and limitations. Like the viruses from which they are derived, VLPs are of interest in both biomedical and materials applications. The encapsulation of protein cargo inside VLPs leads to numerous uses in both fundamental and applied biocatalysis and biomedicine, some of which are discussed herein. The applied science of protein-encapsulating VLPs is emerging as a research field with great potential. Developments in loading control, higher order assembly, and capsid optimization are poised to realize this potential in the near future. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Peptide Self-Assemblies from Unusual α-Sheet Conformations Based on Alternation of d/l Amino Acids.

Peptide self-assembly is a hierarchical process during which secondary structures formed in the initial stages play a critical role in determining the subsequent assembling processes and final structural ordering. Unusual secondary structures hold promise as a source to develop novel supramolecular architectures with unique properties. In this work, we report the design of a new peptide self-assembly strategy based on unusual α-sheet secondary structures. In light of the strong propensity of leucine toward forming helical conformations and its high hydrophobicity, we design two short amphiphilic peptides Ac-LDLLDLK-NH2 and Ac-DLLDLLDK-NH2 with alternating l- and d-form amino acids. Microscopic imaging, neutron scattering, and spectroscopic measurements indicate that the two heterochiral peptides form highly ordered wide nanotubes and helical ribbons with monolayer thickness, in sharp contrast to twisted nanofibrils formed by the homochiral peptide Ac-LLLLK-NH2. Molecular dynamics simulations from monomers to trimers reveal that the two heteropeptides fold into α-sheets instead of β-sheets, which readily pack into tubular architectures in oligomer simulations. Simulated circular dichroism spectra based on α-sheet oligomers validate the proposed α-sheet secondary structures. These results form an important basis for the rational design of higher-order peptide assemblies with novel properties based on unusual α-sheet secondary structures.

Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes

RING-B box-coiled coil (RBCC) domain of promyelocytic leukemia (PML) comprises a zinc finger motif that is targeted by arsenic trioxide (ATO) to treat acute promyelocytic leukemia (APL) pathogenesis. Preliminary evidence suggests that the PML-RBCC has different functional characteristics, but no structural details have been reported despite its importance in differential expression and cell-cycle regulation. Therefore, the recombinant h-PML-RBCC protein was purified to its homogeneity, and characterized for oligomeric behaviour which indicated that RBCC domain exists as a tetramer in solution. Furthermore, nano-DSF and circular-dichroism demonstrated that the tetrameric form preserves its native conformation along with thermal stability (Tm = 83.2 °C). In-silico-based PML-RBCC structure was used to perform the molecular dynamics simulation for 300 ns in the presence of zinc atoms, which demonstrated the differential dynamic of PML-RBCC tetrameric chains. MMPBSA analysis also indicated the role of hydrophobic interactions that favours stable tetrameric structure of PML-RBCC. ATO-induced secondary and tertiary structure changes were observed in PML-RBCC using circular dichroism and fluorescence spectroscopy. Dynamic light scattering and transmission electron microscopy revealed ATO-induced higher-order oligomerization and aggregation of PML-RBCC. The unique oligomeric nature of the h-PML-RBCC protein and its interactions with ATO will help to understand the mechanism of APL pathogenesis and drug resistance.