Modular Assembly of the Bacterial Large Ribosomal Subunit.

The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.

Elucidating the assembly of gas vesicles by systematic protein-protein interaction analysis.

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications.

Discovering optimal kinetic pathways for self-assembly using automatic differentiation.

Macromolecular complexes are often composed of diverse subunits. The self-assembly of these subunits is inherently nonequilibrium and must avoid kinetic traps to achieve high yield over feasible timescales. We show how the kinetics of self-assembly benefits from diversity in subunits because it generates an expansive parameter space that naturally improves the "expressivity" of self-assembly, much like a deeper neural network. By using automatic differentiation algorithms commonly used in deep learning, we searched the parameter spaces of mass-action kinetic models to identify classes of kinetic protocols that mimic biological solutions for productive self-assembly. Our results reveal how high-yield complexes that easily become kinetically trapped in incomplete intermediates can instead be steered by internal design of rate-constants or external and active control of subunits to efficiently assemble. Internal design of a hierarchy of subunit binding rates generates self-assembly that can robustly avoid kinetic traps for all concentrations and energetics, but it places strict constraints on selection of relative rates. External control via subunit titration is more versatile, avoiding kinetic traps for any system without requiring molecular engineering of binding rates, albeit less efficiently and robustly. We derive theoretical expressions for the timescales of kinetic traps, and we demonstrate our optimization method applies not just for design but inference, extracting intersubunit binding rates from observations of yield-vs.-time for a heterotetramer. Overall, we identify optimal kinetic protocols for self-assembly as a powerful mechanism to achieve efficient and high-yield assembly in synthetic systems whether robustness or ease of "designability" is preferred.

Nucleoporin Condensates Drive Nuclear Pore Complex Assembly in Oocytes.

Oocytes stockpile nuclear pore complexes (NPCs) in cytoplasmic membrane sheets called annulate lamellae (AL) in preparation for rapid cell cycles during embryogenesis. Recently, Hampoelz et al. reported that AL-NPC assembly depends on the coordinated formation, transport, and interaction of biomolecular condensates containing distinct sets of nucleoporins.

Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth.

Dynamic assembly and remodeling of actin is critical for many cellular processes during development and stress adaptation. In filamentous fungi and budding yeast, actin cables align in a polarized manner along the mother-to-daughter cell axis, and are essential for the establishment and maintenance of polarity; moreover, they rapidly remodel in response to environmental cues to achieve an optimal system response. A formin at the tip region within a macromolecular complex, called the polarisome, is responsible for driving actin cable polymerization during polarity establishment. This polarisome undergoes dynamic assembly through spatial and temporally regulated interactions between its components. Understanding this process is important to comprehend the tuneable activities of the formin-centered nucleation core, which are regulated through divergent molecular interactions and assembly modes within the polarisome. In this Review, we focus on how intrinsically disordered regions (IDRs) orchestrate the condensation of the polarisome components and the dynamic assembly of the complex. In addition, we address how these components are dynamically distributed in and out of the assembly zone, thereby regulating polarized growth. We also discuss the potential mechanical feedback mechanisms by which the force-induced actin polymerization at the tip of the budding yeast regulates the assembly and function of the polarisome.

A hub-and-spoke nuclear lamina architecture in trypanosomes.

The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration.

The Knowns and Unknowns in Protein-Metabolite Interactions.

Increasing attention has been focused on the study of protein-metabolite interactions (PMI), which play a key role in regulating protein functions and directing an orchestra of cellular processes. The investigation of PMIs is complicated by the fact that many such interactions are extremely short-lived, which requires very high resolution in order to detect them. As in the case of protein-protein interactions, protein-metabolite interactions are still not clearly defined. Existing assays for detecting protein-metabolite interactions have an additional limitation in the form of a limited capacity to identify interacting metabolites. Thus, although recent advances in mass spectrometry allow the routine identification and quantification of thousands of proteins and metabolites today, they still need to be improved to provide a complete inventory of biological molecules, as well as all interactions between them. Multiomic studies aimed at deciphering the implementation of genetic information often end with the analysis of changes in metabolic pathways, as they constitute one of the most informative phenotypic layers. In this approach, the quantity and quality of knowledge about PMIs become vital to establishing the full scope of crosstalk between the proteome and the metabolome in a biological object of interest. In this review, we analyze the current state of investigation into the detection and annotation of protein-metabolite interactions, describe the recent progress in developing associated research methods, and attempt to deconstruct the very term "interaction" to advance the field of interactomics further.

Assembly and Function of the Anthrax Toxin Protein Translocation Complex.

Anthrax toxin is a major virulence factor of Bacillus anthracis, a Gram-positive bacterium which can form highly stable spores that are the causative agents of the disease, anthrax. While chiefly a disease of livestock, spores can be "weaponized" as a bio-terrorist agent, and can be deadly if not recognized and treated early with antibiotics. The intracellular pathways affected by the enzymes are broadly understood and are not discussed here. This chapter focuses on what is known about the assembly of secreted toxins on the host cell surface and how the toxin is delivered into the cytosol. The central component is the "Protective Antigen", which self-oligomerizes and forms complexes with its pay-load, either Lethal Factor or Edema Factor. It binds a host receptor, CMG2, or a close relative, triggering receptor-mediated endocytosis, and forms a remarkably elegant yet powerful machine that delivers toxic enzymes into the cytosol, powered only by the pH gradient across the membrane. We now have atomic structures of most of the starting, intermediate and final assemblies in the infectious process. Together with a major body of biophysical, mutational and biochemical work, these studies reveal a remarkable story of both how toxin assembly is choreographed in time and space.

Cutin extraction and composition determined under differing depolymerisation conditions in cork oak leaves.

INTRODUCTION: Cutin is a biopolyester involved in waterproofing aerial plant organs, including leaves. Cutin quantification and compositional profiling require depolymerisation, namely by methanolysis, but specific protocols are not available. OBJECTIVES: Investigate how different methanolysis conditions regarding catalyst concentration effect cutin depolymerisation and monomer release, to better define protocols for cutin content determination and composition profiling. MATERIAL AND METHODS: Cork oak (Quercus suber) dewaxed leaves were reacted with five sodium methoxide (NaOMe) concentrations. Extracts were analysed: glycerol by high-performance liquid chromatography (HPLC) and long-chain lipids by gas chromatography-mass spectrometry (GC-MS). RESULTS: Cutin was completely removed by 3% NaOMe (8.4% of dewaxed leaves), while mild 0.1% and 0.01% NaOMe methanolysis only depolymerised 14% of total cutin. Reactivity of cutin ester bonds is not homogeneous and glyceridic ester bonds are more easily cleaved, releasing the existing glycerol already under the mildest conditions (0.53% with 0.01% NaOMe and 0.41% with 3% NaOMe). The composition of cutin extracts varies with depolymerisation extent, with easier release of alkanoic acids and alkanols, respectively, 34.9% and 8.8% of total monomers at 0.1% NaOMe, while ω-hydroxyacids (49.3% of total monomers) and α,ω-diacids (9.0% of the monomers) are solubilised under more intensive reactive conditions. CONCLUSION: Cutin of Quercus suber leaves is confirmed as a glyceridic polyester of ω-hydroxyacids and alkanoic acids, with minor content of α,ω-diacids, and including coumarate moieties. The protocol for the determination of cutin content and compositional profiling was established regarding catalyst concentration. The molar composition of cutin suggests a macromolecular assembly based on glycerol linked to lipid oligomeric chains with moderate cross-linking.

Filamentous Bacteriophage Proteins and Assembly.

Filamentous bacteriophages, also known as filamentous bacterial viruses or Inoviruses, have been studied extensively over the years. They are interesting paradigms in structural molecular biology and offer insight into molecular assembly, a process that remains to be fully understood. In this chapter, an overview on filamentous bacteriophages will be provided. In particular, we review the constituent proteins of filamentous bacteriophage and discuss assembly by examining the structure of the major coat protein at various stages of the process. The minor coat proteins will also be briefly reviewed. Structural information provides key snapshots into the dynamic process of assembly.

The Oligomeric State of the Plasma Membrane H⁺-ATPase from Kluyveromyces lactis.

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

Polybivalency and disordered proteins in ordering macromolecular assemblies.

Intrinsically disordered proteins (IDPs) are prevalent in macromolecular assemblies and are thought to mediate protein recognition in complex regulatory processes and signaling pathways. The formation of a polybivalent scaffold is a key process by which IDPs drive early steps in macromolecular assemblies. Three intrinsically disordered proteins, IC, Swallow and Nup159, are core components, respectively, of cytoplasmic dynein, bicoid mRNA localization apparatus, and nuclear pore complexes. In all three systems, the hub protein LC8 recognizes on the IDP, short linear motifs that are fully disordered in the apo form, but adopt a ß-strand when bound to LC8. The IDP/LC8 complex forms a bivalent scaffold primed to bind additional bivalent ligands. Scaffold formation also promotes self-association and/or higher order organization of the IDP components at a site distant from LC8 binding. Rigorous thermodynamic analyses imply that association of additional bivalent ligands is driven by entropic effects where the first binding event is weak but subsequent binding of additional ligands occurs with higher affinity. Here, we review specific examples of macromolecular assemblies in which polybivalency of aligned IDP duplexes not only enhances binding affinity and results in formation of a stable complex but also compensates unfavorable steric and enthalpic interactions. We propose that polybivalent scaffold assembly involving IDPs and LC8-like proteins is a general process in the cell biology of a class of multi-protein structures that are stable yet fine-tuned for diverse cellular requirements.

Higher order structures in purine and pyrimidine metabolism.

The recent discovery of several forms of higher order protein structures in cells has shifted the paradigm of how we think about protein organization and metabolic regulation. These dynamic and controllable protein assemblies, which are composed of dozens or hundreds of copies of an enzyme or related enzymes, have emerged as important players in myriad cellular processes. We are only beginning to appreciate the breadth of function of these types of macromolecular assemblies. These higher order structures, which can be assembled in response to varied cellular stimuli including changing metabolite concentrations or signaling cascades, give the cell the capacity to modulate levels of biomolecules both temporally and spatially. This provides an added level of control with distinct kinetics and unique features that can be harnessed as a subtle, yet powerful regulatory mechanism. Due, in large part, to advances in structural methods, such as crystallography and cryo-electron microscopy, and the advent of super-resolution microscopy techniques, a rapidly increasing number of these higher order structures are being identified and characterized. In this review, we detail what is known about the structure, function and control mechanisms of these mesoscale protein assemblies, with a particular focus on those involved in purine and pyrimidine metabolism. These structures have important implications both for our understanding of fundamental cellular processes and as fertile ground for new targets for drug discovery and development.

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing.

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed "ruler" protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a "ball-and-chain" architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein's N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.

A gold electrode modified with a nanoparticulate film composed of a conducting copolymer for ultrasensitive voltammetric sensing of hydrogen peroxide.

A film consisting of poly(γ-glutamic acid) modified with 3-aminothiophene (ATh-γ-PGA) was prepared by macromolecular self-assembly and electropolymerization. ATh-γ-PGA is amphiphilic and electrically conductive. The copolymers undergo self-assembly to form nanoparticles (NPs) on decreasing the pH value of an aqueous solution. A conducting film of NPs was formed on the surface of a gold electrode by casting the ATh-γ-PGA NPs and subsequently electropolymerizing the thiophene units. Next, horseradish peroxidase and Nafion were cast onto the film to obtain an enzymatic biosensor for H2O2. Due to the electropolymerization step, a cross-conjugated polymer network is created that improves electron transfer rates and thus enhances the response. This endows the biosensor with high sensitivity. Two linear ranges are present, the first ranging from 1 × 10-11 to 1 × 10-8 mol·L-1, and the second from 1 × 10-8 to 1 × 10-5 mol·L-1. The detection limit is as low as 3 × 10-12 mol·L-1. The sensor is stable, repeatable, and was successfully applied to the determination of H2O2 in a commercial disinfecting solution. Graphical abstract Preparation of a conducting nanoparticle (NP) film on the gold electrode (GE) by self-assembly of poly(γ-glutamic acid) that was modified with electroactive 3-aminothiophene (ATh-γ-PGA). It served as a platform for the fabricationof an ultrasensitive voltammetric enzyme-based biosensor for H2O2.

Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

Two-state dynamics of the SH3-SH2 tandem of Abl kinase and the allosteric role of the N-cap.

The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3-SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3-SH2 connector, which involve a phosphorylation site. We also show that the SH3-SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3-SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis.

Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane ß-barrel translocation domain (TD), a ß-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (ß12-13 loop) was found to facilitate pore opening. Mutation of the ß12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.

Large oligomeric complex structures can be computationally assembled by efficiently combining docked interfaces.

Macromolecular oligomeric assemblies are involved in many biochemical processes of living organisms. The benefits of such assemblies in crowded cellular environments include increased reaction rates, efficient feedback regulation, cooperativity and protective functions. However, an atom-level structural determination of large assemblies is challenging due to the size of the complex and the difference in binding affinities of the involved proteins. In this study, we propose a novel combinatorial greedy algorithm for assembling large oligomeric complexes from information on the approximate position of interaction interfaces of pairs of monomers in the complex. Prior information on complex symmetry is not required but rather the symmetry is inferred during assembly. We implement an efficient geometric score, the transformation match score, that bypasses the model ranking problems of state-of-the-art scoring functions by scoring the similarity between the inferred dimers of the same monomer simultaneously with different binding partners in a (sub)complex with a set of pregenerated docking poses. We compiled a diverse benchmark set of 308 homo and heteromeric complexes containing 6 to 60 monomers. To explore the applicability of the method, we considered 48 sets of parameters and selected those three sets of parameters, for which the algorithm can correctly reconstruct the maximum number, namely 252 complexes (81.8%) in, at least one of the respective three runs. The crossvalidation coverage, that is, the mean fraction of correctly reconstructed benchmark complexes during crossvalidation, was 78.1%, which demonstrates the ability of the presented method to correctly reconstruct topology of a large variety of biological complexes.

ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions.

Asymmetric ASH1 mRNA transport during mitosis of budding yeast constitutes one of the best-studied examples of mRNA localization. Recently, 2 studies used in vitro motility assays to prove that motile ASH1 mRNA-transport complexes can be reconstituted entirely from recombinant factors. Both studies, however, differed in their conclusions on whether cargo RNA itself is required for particle assembly and thus activation of directional transport. Here we provide direct evidence that stable complexes do assemble in absence of RNA at physiologic conditions and even at ionic strengths above cellular levels. These results directly confirm the previous notion that the ASH1 transport machinery is not activated by the cargo RNA itself, but rather through protein-protein interactions.