Flexible, symmetry-directed approach to assembling protein cages

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.

Download Full-text

Coiled-Coils: The Molecular Zippers that Self-Assemble Protein Nanostructures

International Journal of Molecular Sciences ◽

10.3390/ijms21103584 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3584 ◽

Cited By ~ 1

Author(s):

Won Min Park

Keyword(s):

Modular Design ◽

Coiled Coil ◽

Molecular Complexes ◽

Building Blocks ◽

Structural Features ◽

Coiled Coils ◽

Design Strategies ◽

Protein Motifs ◽

Self Assembling ◽

Current Design

Coiled-coils, the bundles of intertwined helical protein motifs, have drawn much attention as versatile molecular toolkits. Because of programmable interaction specificity and affinity as well as well-established sequence-to-structure relationships, coiled-coils have been used as subunits that self-assemble various molecular complexes in a range of fields. In this review, I describe recent advances in the field of protein nanotechnology, with a focus on programming assembly of protein nanostructures using coiled-coil modules. Modular design approaches to converting the helical motifs into self-assembling building blocks are described, followed by a discussion on the molecular basis and principles underlying the modular designs. This review also provides a summary of recently developed nanostructures with a variety of structural features, which are in categories of unbounded nanostructures, discrete nanoparticles, and well-defined origami nanostructures. Challenges existing in current design strategies, as well as desired improvements for controls over material properties and functionalities for applications, are also provided.

Download Full-text

Targeting viral genome synthesis as broad-spectrum approach against RNA virus infections

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206620976786 ◽

2020 ◽

Vol 28 ◽

pp. 204020662097678

Author(s):

Johanna Huchting

Keyword(s):

Broad Spectrum ◽

Viral Genome ◽

De Novo ◽

Rna Viruses ◽

Rna Virus ◽

Antiviral Drug ◽

Building Blocks ◽

Structural Features ◽

Pathogen Transmission ◽

Nucleotide Synthesis

Zoonotic spillover, i.e. pathogen transmission from animal to human, has repeatedly introduced RNA viruses into the human population. In some cases, where these viruses were then efficiently transmitted between humans, they caused large disease outbreaks such as the 1918 flu pandemic or, more recently, outbreaks of Ebola and Coronavirus disease. These examples demonstrate that RNA viruses pose an immense burden on individual and public health with outbreaks threatening the economy and social cohesion within and across borders. And while emerging RNA viruses are introduced more frequently as human activities increasingly disrupt wild-life eco-systems, therapeutic or preventative medicines satisfying the “one drug-multiple bugs”-aim are unavailable. As one central aspect of preparedness efforts, this review digs into the development of broadly acting antivirals via targeting viral genome synthesis with host- or virus-directed drugs centering around nucleotides, the genomes’ universal building blocks. Following the first strategy, selected examples of host de novo nucleotide synthesis inhibitors are presented that ultimately interfere with viral nucleic acid synthesis, with ribavirin being the most prominent and widely used example. For directly targeting the viral polymerase, nucleoside and nucleotide analogues (NNAs) have long been at the core of antiviral drug development and this review illustrates different molecular strategies by which NNAs inhibit viral infection. Highlighting well-known as well as recent, clinically promising compounds, structural features and mechanistic details that may confer broad-spectrum activity are discussed. The final part addresses limitations of NNAs for clinical development such as low efficacy or mitochondrial toxicity and illustrates strategies to overcome these.

Download Full-text

De novo assembly and delivery to mouse cells of a 101 kb functional human gene

Genetics ◽

10.1093/genetics/iyab038 ◽

2021 ◽

Author(s):

Leslie A Mitchell ◽

Laura H McCulloch ◽

Sudarshan Pinglay ◽

Henri Berger ◽

Nazario Bosco ◽

...

Keyword(s):

Dna Sequences ◽

Large Scale ◽

De Novo ◽

Human Gene ◽

Embryonic Stem ◽

Building Blocks ◽

Functional Study ◽

Dna Assembly ◽

Functional Evaluation ◽

Mouse Cells

Abstract Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

Download Full-text

Multi-scale structural analysis of proteins by deep semantic segmentation

Bioinformatics ◽

10.1093/bioinformatics/btz650 ◽

2019 ◽

Vol 36 (6) ◽

pp. 1740-1749 ◽

Cited By ~ 2

Author(s):

Raphael R Eguchi ◽

Po-Ssu Huang

Keyword(s):

Protein Design ◽

Large Scale ◽

De Novo ◽

Protein Structures ◽

Semantic Segmentation ◽

Structural Features ◽

Supplementary Information ◽

Structural Prediction ◽

Structure Accuracy ◽

High Level

Abstract Motivation Recent advances in computational methods have facilitated large-scale sampling of protein structures, leading to breakthroughs in protein structural prediction and enabling de novo protein design. Establishing methods to identify candidate structures that can lead to native folds or designable structures remains a challenge, since few existing metrics capture high-level structural features such as architectures, folds and conformity to conserved structural motifs. Convolutional Neural Networks (CNNs) have been successfully used in semantic segmentation—a subfield of image classification in which a class label is predicted for every pixel. Here, we apply semantic segmentation to protein structures as a novel strategy for fold identification and structure quality assessment. Results We train a CNN that assigns each residue in a multi-domain protein to one of 38 architecture classes designated by the CATH database. Our model achieves a high per-residue accuracy of 90.8% on the test set (95.0% average per-class accuracy; 87.8% average per-structure accuracy). We demonstrate that individual class probabilities can be used as a metric that indicates the degree to which a randomly generated structure assumes a specific fold, as well as a metric that highlights non-conformative regions of a protein belonging to a known class. These capabilities yield a powerful tool for guiding structural sampling for both structural prediction and design. Availability and implementation The trained classifier network, parser network, and entropy calculation scripts are available for download at https://git.io/fp6bd, with detailed usage instructions provided at the download page. A step-by-step tutorial for setup is provided at https://goo.gl/e8GB2S. All Rosetta commands, RosettaRemodel blueprints, and predictions for all datasets used in the study are available in the Supplementary Information. Supplementary information Supplementary data are available at Bioinformatics online.

Download Full-text

De Novo Designed Peptide and Protein Hairpins Self-assemble into Sheets and Nanoparticles

10.1101/2020.08.14.251462 ◽

2020 ◽

Author(s):

Johanna M. Galloway ◽

Harriet E. V. Bray ◽

Deborah K. Shoemark ◽

Lorna R. Hodgson ◽

Jennifer Coombs ◽

...

Keyword(s):

Secondary Structure ◽

Disulfide Bond ◽

Self Assembly ◽

De Novo ◽

Coiled Coil ◽

Building Blocks ◽

Chemical Modifications ◽

Peptide System ◽

And Control ◽

Similar Peptide

AbstractThe design and assembly of peptide based materials has advanced considerably, leading to a variety of fibrous, sheet and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here we present a de novo peptide system that forms nanoparticles or sheets depending on the strategic placement of a ‘disulfide pin’ between two elements of secondary structure that drive self-assembly. Specifically, we join homodimerizing and homotrimerizing de novo coiled-coil α-helices with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling and advanced microscopy show that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.

Download Full-text

Coiled Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability

10.1101/316331 ◽

2018 ◽

Author(s):

Ajitha S. Cristie-David ◽

Junjie Chen ◽

Derek B. Nowak ◽

Sung I. Park ◽

Mark M. Banaszak Holl ◽

...

Keyword(s):

De Novo ◽

Guanidine Hydrochloride ◽

Coiled Coil ◽

Higher Order ◽

Order Structure ◽

Icosahedral Symmetry ◽

Force Microscopy ◽

Protein Cage ◽

Negative Stain ◽

Icosahedral Viruses

AbstractThe organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Here we describe the symmetry-directed design of a hollow protein cage with dimensions similar to those of many icosahedral viruses. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain and cryo EM, and by atomic force and IR-photo-induced force microscopy established that Ico8 assembles into a flexible hollow cage with comprising 60-subunits with overall icosahedral geometry. Unexpectedly, the cages were found to encapsulate DNA, even though neither protein component binds nucleic acids on its own. Notably, the cages formed by Ico8 proved to be extremely stable towards thermal and chemical denaturation: whereas TriEst was unfolded by heating (Tm ~75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The encapsulation of DNA and increased stability of the cages are new properties that emerge from the higher order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Download Full-text

Design of two-stranded and three-stranded coiled-coil peptides

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0048 ◽

1995 ◽

Vol 348 (1323) ◽

pp. 81-88 ◽

Cited By ~ 52

Keyword(s):

Protein Design ◽

De Novo ◽

Coiled Coil ◽

Structural Features ◽

Coiled Coils ◽

General Sequence ◽

The Third ◽

Cooperative Equilibrium ◽

High Concentrations ◽

Propensity Scale

The structural features required for the formation of two- versus three-stranded coiled coils have been explored using de novo protein design. Peptides with leucine at the ‘a’ and ‘d’ positions of a coiled-coil (general sequence: Leu a Xaa b Xaa c Leu d Glu e Xaa f Lys g ) exist in a non-cooperative equilibrium between unstructured monomers and helical dimers and helical trimers. Substituting valine into each ‘a’ position produces peptides which still form trimers at high concentrations, whereas substitution of a single asparagine at the ‘a’ position of the third heptad yields a dimer. During the course of this work, we also re-investigated a helical propensity scale derived using a series of coiled-coil peptides previously believed to exist in a monomer-dimer equilibrium (O’Neil & DeGrado 1990). Detailed analysis of the concentration dependence of ellipticity at 222 nm reveals that they exist in a non-cooperative monomer-dimer-trimer equilibrium. However, the concentration of trimer near the midpoint of the concentration-dependent transition is small, so the previously determined values of ΔΔG α using the approximate monomer-dimer scheme are indistinguishable from the values obtained employing the complete monomer-dimer-trimer equilibrium.

Download Full-text

New insight on the structural diversity of holothurian fucosylated chondroitin sulfates

Pure and Applied Chemistry ◽

10.1515/pac-2018-1211 ◽

2019 ◽

Vol 91 (7) ◽

pp. 1065-1071 ◽

Cited By ~ 9

Author(s):

Nadezhda E. Ustyuzhanina ◽

Maria I. Bilan ◽

Nikolay E. Nifantiev ◽

Anatolii I. Usov

Keyword(s):

Biological Effect ◽

Wide Spectrum ◽

Structural Diversity ◽

Building Blocks ◽

Structural Features ◽

Biological Properties ◽

Structural Variations ◽

Chondroitin Sulfates ◽

Nmr Data ◽

Species Specific

AbstractFucosylated chondroitin sulfates (FCS) are unique glycosaminoglycans isolated from body walls of sea cucumbers (holothuria). These biopolymers are composed of a chondroitin core [→4)-β-D-GlcA-(1→3)-β-D-GalNAc-(1→]nbearing fucosyl branches and sulfate groups. Structural variations of FCS are species specific and depend on type, amount and position of branches, as well as on degree and pattern of sulfation of a backbone and branches. A wide spectrum of biological properties was determined for these polysaccharides including anticoagulant, antithrombotic, antitumor, anti-inflammatory activities. Structural features of FCS influence significantly on their biological effect. In this review recent data about structural variations within holothurian FCS are summarized. The NMR data of the key building blocks are presented, which may be used for the analysis of new FCS.

Download Full-text

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

10.26434/chemrxiv.11663337.v3 ◽

2020 ◽

Author(s):

Salvador Guardiola ◽

Monica Varese ◽

Xavier Roig ◽

Jesús Garcia ◽

Ernest Giralt

Keyword(s):

Protein Interactions ◽

Cyclic Peptides ◽

General Framework ◽

Large Scale ◽

De Novo ◽

Inhibitory Effect ◽

Original Text ◽

Protein Protein Interactions ◽

Retraction Notice ◽

Pharmaceutical Properties

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above. ------------------------------------------------------------------------ Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

Download Full-text