scholarly journals Hot-spots and their contribution to the self-assembly of the viral capsid: in-vitro validation

2019 ◽  
Author(s):  
Alejandra Gabriela Valdez-Lara ◽  
Mariana Andrade-Medina ◽  
Josué Alejandro Alemán-Vilis ◽  
Aldo Adrián Pérez-Montoya ◽  
Nayely Pineda-Aguilar ◽  
...  
Keyword(s):  
Self Assembly ◽  
Hot Spots ◽  
Point Mutations ◽  
The Self ◽  
Viral Capsid ◽  
Single Mutation ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Interface Residues

AbstractThe viral capsid is a macromolecular complex formed by self-assembled proteins (CPs) which, in many cases, are biopolymers with an identical amino acid sequence. Specific CP-CP interactions drive the capsid self-assembly process. However, it is believed that only a small set of protein-protein interface residues significantly contribute to the formation of the capsid; the so-called “hot-spots”. Here, we investigate the effect of in-vitro point-mutations on the Bromoviridae family structure-conserved interface residues of the icosahedral Cowpea Chlorotic Mottle Virus, previously hypothesized as hot-spots. We study the self-assembly of those mutated recombinant CPs for the formation of capsids by Thermal Shift Assay (TSA). We show that the TSA biophysical technique is a reliable way to characterize capsid assembly. Our results show that point-mutations on non-conserved interface residues produce capsids indistinguishable from the wild-type. In contrast, a single mutation on structure-conserved residues E176 or V189 prevents the formation of the capsid while maintaining the tertiary fold of the CP. Our findings provide experimental evidence of the in-silico conservation-based hot-spot prediction accuracy. As a whole, our methodology provides a framework that could aid in the rational development of molecules to inhibit virus formation, or advance capsid bioengineering to design for their stability, function and applications.

scholarly journals Hot Spots and Their Contribution to the Self-Assembly of the Viral Capsid: In Silico Prediction and Analysis

2019 ◽  
Vol 20 (23) ◽  
pp. 5966 ◽  
Author(s):  
Armando Díaz-Valle ◽  
José Marcos Falcón-González ◽  
Mauricio Carrillo-Tripp
Keyword(s):  
Free Energy ◽  
Self Assembly ◽  
Hot Spots ◽  
Binding Free Energy ◽  
Hot Spot ◽  
Free Energy Calculations ◽  
The Self ◽  
Viral Capsid ◽  
Macromolecular Complex ◽  
Single Mutation

The viral capsid is a macromolecular complex formed by a defined number of self-assembled proteins, which, in many cases, are biopolymers with an identical amino acid sequence. Specific protein–protein interactions (PPI) drive the capsid self-assembly process, leading to several distinct protein interfaces. Following the PPI hot spot hypothesis, we present a conservation-based methodology to identify those interface residues hypothesized to be crucial elements on the self-assembly and thermodynamic stability of the capsid. We validate the predictions through a rigorous physical framework which integrates molecular dynamics simulations and free energy calculations by Umbrella sampling and the potential of mean force using an all-atom molecular representation of the capsid proteins of an icosahedral virus in an explicit solvent. Our results show that a single mutation in any of the structure-conserved hot spots significantly perturbs the quaternary protein–protein interaction, decreasing the absolute value of the binding free energy, without altering the protein’s secondary nor tertiary structure. Our conservation-based hot spot prediction methodology can lead to strategies to rationally modulate the capsid’s thermodynamic properties.

scholarly journals Hot-spots and their contribution to the self-assembly of the viral capsid: in-silico prediction and analysis

2019 ◽  
Author(s):  
Armando Díaz-Valle ◽  
José Marcos Falcón-González ◽  
Mauricio Carrillo-Tripp
Keyword(s):  
Free Energy ◽  
Self Assembly ◽  
Hot Spots ◽  
Binding Free Energy ◽  
Free Energy Calculations ◽  
The Self ◽  
Atomic Scale ◽  
Viral Capsid ◽  
Macromolecular Complex ◽  
Single Mutation

AbstractIn order to rationally design biopolymers that mimic biological functions, first, we need to elucidate the molecular mechanisms followed by nature. For example, the viral capsid is a macromolecular complex formed by self-assembled proteins which, in many cases, are biopolymers with an identical amino acid sequence. Specific protein-protein interactions drive the capsid self-assembly process, leading to several distinct protein interfaces. Following the hot-spot hypothesis, we propose a conservation-based methodology to identify those interface residues that are crucial elements on the self-assembly and thermodynamic stability of the capsid. We validate our predictions by computational free energy calculations using an atomic-scale molecular model of an icosahedral virus. Our results show that a single mutation in any of the hot-spots significantly perturbs the quaternary interaction, decreasing the absolute value of the binding free energy, without altering the tertiary structure. Our methodology can lead to a strategy to rationally modulate the capsid’s thermodynamic properties.

scholarly journals Synergistic Effects of Mutations and Nanoparticle Templating in the Self-Assembly of Cowpea Chlorotic Mottle Virus Capsids

Nano Letters ◽  
2009 ◽  
Vol 9 (1) ◽  
pp. 393-398 ◽  
Author(s):  
Stella E. Aniagyei ◽  
Chelsea J. Kennedy ◽  
Barry Stein ◽  
Deborah A. Willits ◽  
Trevor Douglas ◽  
...  
Keyword(s):  
Self Assembly ◽  
Synergistic Effects ◽  
The Self ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Virus Capsids

Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins inPichia pastorisand Encapsulation of Fluorescent Myoglobin

2011 ◽  
Vol 1317 ◽  
Author(s):  
Yuanzheng Wu ◽  
Hetong Yang ◽  
Hyun-Jae Shin
Keyword(s):  
Coat Protein ◽  
Large Scale ◽  
Materials Science ◽  
Cesium Chloride ◽  
Size Exclusion ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

Abstract:Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over 40 years and now is considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self assemblein vitrointo virus-like particles (VLPs) or capsids makes an ideal reaction vessel for nanomaterial synthesis and entrapment. Here we report expression of codon optimized CCMV coat protein inPichia pastorisand production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference ofP. pastorisand cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed intoP. pastorisGS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentation CCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified by modified PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and then analyzed by size exclusion fast performance liquid chromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin was used as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopy showed that inclusion of myoglobin had occurred. The results indicated the production of CCMV capsids byP. pastorisfermentation now available for utilization in pharmacology or nanotechnology fields.

scholarly journals Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

2018 ◽  
Author(s):  
Ailís O’Carroll ◽  
Brieuc Chauvin ◽  
James Brown ◽  
Ava Meagher ◽  
Joanne Coyle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Full Length ◽  
Binary Response ◽  
Death Domain ◽  
Tir Domain ◽  
The Impact

AbstractA novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a “seed” that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital ‘all-or-none’ responses and the behaviour of the oncogenic mutation of MyD88.

scholarly journals Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Michal Wieczorek ◽  
Shih-Chieh Ti ◽  
Linas Urnavicius ◽  
Kelly R. Molloy ◽  
Amol Aher ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
The Self ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Partial Cone ◽  
Ring Complex ◽  
Microtubule Networks ◽  
And Function

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.

scholarly journals Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

2006 ◽  
Vol 80 (7) ◽  
pp. 3582-3591 ◽  
Author(s):  
Jeffrey A. Speir ◽  
Brian Bothner ◽  
Chunxu Qu ◽  
Deborah A. Willits ◽  
Mark J. Young ◽  
...  
Keyword(s):  
Critical Role ◽  
Local Symmetry ◽  
High Salt ◽  
Direct Result ◽  
Wild Type ◽  
Particle Structure ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

ABSTRACT Structural transitions in viral capsids play a critical role in the virus life cycle, including assembly, disassembly, and release of the packaged nucleic acid. Cowpea chlorotic mottle virus (CCMV) undergoes a well-studied reversible structural expansion in vitro in which the capsid expands by 10%. The swollen form of the particle can be completely disassembled by increasing the salt concentration to 1 M. Remarkably, a single-residue mutant of the CCMV N-terminal arm, K42R, is not susceptible to dissociation in high salt (salt-stable CCMV [SS-CCMV]) and retains 70% of wild-type infectivity. We present the combined structural and biophysical basis for the chemical stability and viability of the SS-CCMV particles. A 2.7-Å resolution crystal structure of the SS-CCMV capsid shows an addition of 660 new intersubunit interactions per particle at the center of the 20 hexameric capsomeres, which are a direct result of the K42R mutation. Protease-based mapping experiments of intact particles demonstrate that both the swollen and closed forms of the wild-type and SS-CCMV particles have highly dynamic N-terminal regions, yet the SS-CCMV particles are more resistant to degradation. Thus, the increase in SS-CCMV particle stability is a result of concentrated tethering of subunits at a local symmetry interface (i.e., quasi-sixfold axes) that does not interfere with the function of other key symmetry interfaces (i.e., fivefold, twofold, quasi-threefold axes). The result is a particle that is still dynamic but insensitive to high salt due to a new series of bonds that are resistant to high ionic strength and preserve the overall particle structure.

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