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The authors report the structure of the assembled membrane spanning ring forming proteins of the Salmonella Typhimurium injectisome basal body, including the first atomic structure of a member of the secretin family of outer-membrane pores. This study is a tour de force of cryo-electron microscopy, presenting direct experimental atomic structures of the assembled inner-membrane rings as well as of the outer-membrane secretin of the basal body of the Salmonella enterica serovar Typhimurium type III secretion (T3S) injectisome. The T3S injectisome is a syringe-shaped macromolecular complex that facilitates the direct delivery of bacterial virulence effectors to the host-cell cytoplasm. This is the first high-resolution structural characterization of the basal body in the assembled state and could usher in a new era of research into the mechanisms of T3S system. The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm1. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes2,3,4,5. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 A and 3.6 A resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.

Summary Members of the YidC/Oxa1/Alb3 family universally facilitate membrane protein biogenesis, via mechanisms that have thus far remained unclear. Here, we investigated two crucial functional aspects: the interaction of YidC with ribosome:nascent chain complexes (RNCs) and the structural dynamics of RNC-bound YidC in nanodiscs. We observed that a fully exposed nascent transmembrane domain (TMD) is required for high-affinity YidC:RNC interactions, while weaker binding may already occur at earlier stages of translation. YidC efficiently catalyzed the membrane insertion of nascent TMDs in both fluid and gel phase membranes. Cryo-electron microscopy and fluorescence analysis revealed a conformational change in YidC upon nascent chain insertion: the essential TMDs 2 and 3 of YidC were tilted, while the amphipathic helix EH1 relocated into the hydrophobic core of the membrane. We suggest that EH1 serves as a mechanical lever, facilitating a coordinated movement of YidC TMDs to trigger the release of nascent chains into the membrane., Graphical Abstract, Highlights • YidC:ribosome assembly strongly depends on the nascent chain length • Gel phase membranes allow the transient YidC:nascent chain complex to be stabilized • Single-particle cryo-EM reveals the structure of ribosome-bound YidC in nanodiscs • YidC undergoes a conformational change upon co-translational substrate insertion, Kedrov et al. use a combination of biochemical, biophysical, and structural approaches to study the insertase YidC in its native lipid environment upon interaction with ribosomes. The results describe how YidC recognizes translating ribosomes and changes its conformation upon nascent chain insertion.

A,D-substituted cyclodextrin (CDX) substituted on their primary rim side are ideal scaffolds for the macromolecular assembly and formation of templated structures. Their substitution can be achieved through various reactions. However, the use of the well-known Huisgen reaction in this context is under-reported. We present here results of the synthesis of model conjugates formed between CDX and representative peptides by click chemistry. Notably, bis-conjugation of peptides onto a unique scaffold promotes α-helix formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Protein-protein interactions play a very important role in the process of cellular functionality. Intricate details about the interactions between the proteins in a macromolecular assembly are important to understand the function and significance of protein complexes. We are reporting about a database of protein-protein interactions in huge macromolecular assemblies (PIMADb) that records the intrinsic details of 189,532 interchain interactions in 40,049 complexes from the Protein Data Bank. These details include the results of the quantification and analysis of all the interactions in the complex. The availability of interprotomer interaction networks can enable the design of point mutation experiments. PIMADb can be accessed from the URL: http://caps.ncbs.res.in/pimadb.

Lack of structure is often an essential functional feature of protein domains. The coordination of macromolecular assemblies in DNA repair pathways is yet another task disordered protein regions are highly implicated in. Here I review the available experimental and computational data and within this context discuss the functional role of structure and disorder in one of the essential scaffolding proteins in the nucleotide excision repair (NER) pathway, namely Xeroderma pigmentosum complementation group A (XPA). From the analysis of the current knowledge, in addition to protein–protein docking and secondary structure prediction results presented for the first time herein, a mechanistic framework emerges, where XPA builds the NER pre-incision complex in a modular fashion, as “beads on a string”, where the protein–protein interaction “beads”, or modules, are interconnected by disordered link regions. This architecture is ideal to avoid the expected steric hindrance constraints of the DNA expanded bubble. Finally, the role of the XPA structural disorder in binding affinity modulation and in the sequential binding of NER core factors in the pre-incision complex is also discussed.

Chromatin-remodeling enzymes perform the formidable task of reorganizing the structure of a stable macromolecular assembly, the nucleosome. Recently published work demonstrates that the SNF2H chromatin remodeler distorts the histone octamer structure upon binding to the nucleosome, then taps into this induced plasticity to productively achieve nucleosome sliding.

Biological macromolecules must fold into native structures to gain functional activities. In living cells, proteins called molecular chaperones mediate productive folding by preventing undesired interactions and aggregation and by facilitating refolding of misfolded macromolecules into their bioactive forms. Inspired by natural molecular chaperones, artificial chaperones that mimic some features of their biological counterparts have been designed. This review describes recent progress in the development of artificial chaperones and their promising applications in enhancing macromolecular assembly of proteins, polypeptides, and nucleic acids.

Chromatin is the macromolecular assembly containing the cell’s genetic information, and its architectural conformation facilitates accessibility to activation sites and thus gene expression. We have developed an analytical framework to quantify chromatin structure with spectral microscopy. Chromatin structure can be described as a mass fractal, with packing scaling D up to specific genomic length scales. Considering various system geometries, we established a model to measure D with the interferometric technique partial wave spectroscopy (PWS) and validated the analysis using finite difference time domain to simulate the PWS system. Calculations of D were consistent with ground truth electron microscopy measurements, enabling a high-throughput, label-free approach to quantifying chromatin structure in the nanometer length scale regime.

Made available in DSpace on 2019-10-06T16:00:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Supramolecular gels are soft materials formed mainly by low molecular weight units held together by intermolecular interactions. Stabilizing these kinds of materials is quite a challenge due to the influence of multiple factors interfering with the integrity of the supramolecular structure. In our previous studies, we have shown that the aminocarbohydrate meglumine (MEG) interacts with organic acids by ion-pairing leading to the formation of MEG–carboxylate adducts. These adducts undergo supramolecular polymerization by heat treatment, but the macromolecular assembly was stable for a short period due to hydrogen bond (H-bond) breakup. Herein, we attempt to study the influence of hydrophobic building blocks on the formation of these compounds aiming to stabilize H-bonds to produce polymerizable supra-amphiphiles in water. Oleic acid and stearic acid are two analogous fatty acids differing only in the presence of unsaturation that were used in our studies. Results demonstrated that the presence of unsaturation hinders gelation in water by interfering with the self-assembly behavior of supra-amphiphiles. Thus, unsaturated supra-amphiphiles behave like traditional surfactants and gelify water at high concentrations (above 30% w/w). On the other hand, supramolecular gels with a polymer-like behavior could be produced with a saturated supra-amphiphile in water (above 4% w/w). The material was characterized by a lamellar arrangement that facilitates the alignment of H-bonds necessary to stabilize the self-assembled structure. These results have pivotal importance on the design of polymerizable supra-amphiphiles and demonstrate that the double bond of hydrophobic building blocks is an important design factor to be considered by scientists studying similar materials. Department of Chemistry Federal University of Sergipe (UFS), Campus (Itabaiana), Av. Vereador Olimpio Grande s/n School of Pharmaceutical Sciences São Paulo State University (UNESP), Campus (Araraquara), Rodovia Araraquara-Jau Km 1 Analytical Chemistry Department São Paulo State University IQ/UNESP, Professor Francisco Degni Street 55 School of Pharmaceutical Sciences São Paulo State University (UNESP), Campus (Araraquara), Rodovia Araraquara-Jau Km 1 Analytical Chemistry Department São Paulo State University IQ/UNESP, Professor Francisco Degni Street 55

The expanding toolkit of structural biology, with further advances in the technologies associated with cryoEM, synchrotrons and XFELs, and the ease of their use, should continue to enable the wider community to address more complicated and demanding scientific questions, ensuring the ‘pole position’ of structural biology., Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. The combination of results from X-ray free-electron lasers (XFELs), modern synchrotron crystallographic beamlines and cryo-electron microscopy (cryoEM) is proving to be particularly powerful. The highly brilliant undulator beamlines at modern synchrotron facilities have empowered the crystallographic revolution of high-throughput structure determination at high resolution. The brilliance of the X-rays at these crystallographic beamlines has enabled this to be achieved using microcrystals, but at the expense of an increased absorbed X-ray dose and a consequent vulnerability to radiation-induced changes. The advent of serial femtosecond crystallography (SFX) with X-ray free-electron lasers provides a new opportunity in which damage-free structures can be obtained from much smaller crystals (2 µm) and more complex macromolecules, including membrane proteins and multi-protein complexes. For redox enzymes, SFX provides a unique opportunity by providing damage-free structures at both cryogenic and ambient temperatures. The promise of being able to visualize macromolecular structures and complexes at high resolution without the need for crystals using X-rays has remained a dream, but recent technological advancements in cryoEM have made this come true and hardly a month goes by when the structure of a new/novel macromolecular assembly is not revealed. The uniqueness of cryoEM in providing structural information for multi-protein complexes, particularly membrane proteins, has been demonstrated by examples such as respirasomes. The synergistic use of cryoEM and crystallography in lead-compound optimization is highlighted by the example of the visualization of antimalarial compounds in cytochrome bc 1. In this short review, using some recent examples including our own work, we share the excitement of these powerful structural biology methods.

In concentrated macromolecular solutions, weak physical interactions control the solution behavior including particle size distribution, aggregation, liquid-liquid phase separation, or crystallization. This is central to many fields ranging from colloid chemistry to cell biology and pharmaceutical protein engineering. Unfortunately, it is very difficult to determine macromolecular assembly states and polydispersity at high concentrations in solution, since all motion is coupled through long-range hydrodynamic, electrostatic, steric, and other interactions, and scattering techniques report on the solution structure when average interparticle distances are comparable to macromolecular dimensions. Here we present a sedimentation velocity technique that, for the first time, can resolve macromolecular size distributions at high concentrations, by simultaneously accounting for average mutual hydrodynamic and thermodynamic interactions. It offers high resolution and sensitivity of protein solutions up to 50 mg/ml, extending studies of macromolecular solution state closer to the concentration range of therapeutic formulations, serum, or intracellular conditions., Many aspects of concentrated macromolecular solutions, such as encountered in cytosol or in pharmaceutical formulations, are dependent on particle size distributions and weak intermolecular interactions. Here, the authors exploit hydrodynamic separation in the centrifugal field to measure both.

Ion mobility-mass spectrometry (IM-MS) allows the separation of isomeric and isobaric species on the basis of their size, shape, and charge. The fast separation timescale (ms) and high sensitivity of these measurements make IM-MS an ideally suitable method for monitoring changes in macromolecular structure, such as those occurring in interconverting terpyridine-based metallosupramolecular self-assemblies. IM-MS is used to verify the elemental composition (size) and architecture (shape) of the self-assembled products. Additionally, this article demonstrates its applicability to the elucidation of concentration-driven association-dissociation (fusion-fission) equilibria between isobaric structures. IM-MS enables both quantitative separation and identification of the interconverting complexes as well as derivation of the corresponding equilibrium constants (i.e., thermodynamic information) from extracted IM-MS abundance data.

Background: First developed in the 1990’s at the National Synchrotron Light Source, xray synchrotron footprinting is an ideal technique for the analysis of solution-state structure and dynamics of macromolecules. Hydroxyl radicals generated in aqueous samples by intense x-ray beams serve as fine probes of solvent accessibility, rapidly and irreversibly reacting with solvent exposed residues to provide a “snapshot” of the sample state at the time of exposure. Over the last few decades, improvements in instrumentation to expand the technology have continuously pushed the boundaries of biological systems that can be studied using the technique. Conclusion: Dedicated synchrotron beamlines provide important resources for examining fundamental biological mechanisms of folding, ligand binding, catalysis, transcription, translation, and macromolecular assembly. The legacy of synchrotron footprinting at NSLS has led to significant improvement in our understanding of many biological systems, from identifying key structural components in enzymes and transporters to in vivo studies of ribosome assembly. This work continues at the XFP (17-BM) beamline at NSLS-II and facilities at ALS, which are currently accepting proposals for use.

Component C6 is a single-chain plasma glycoprotein of Mr ∼ 108,000 that participates in the last phase of complement activation, namely the formation of the membrane attack complex. This macromolecular assembly consists of C5b through C9 and is a transmembrane pore with a channel diameter of ∼100 A. The polypeptide chain of C6 consists of a single domain of about 250 amino acids referred to as the MACPF along with nine cysteine-rich modules (34–77 amino acids). C6 has two binding sites for C5b; one is reversibly mediated by the C-terminal modules and the other is irreversibly formed by a linker between those modules and the rest of the protein. To C5b6 is added C7, C8 and C9 fashioning a circular complex. The gene for C6 is closely linked to that of C7 on chromosome 5. The importance of C6 for the immune system is demonstrated by rare genetic deficiencies of C6 that render patients susceptible to neisserial infections.

Bacterial chemoreceptors form a highly ordered array in concert with the CheA kinase and the CheW coupling protein. The precise architecture of the array is responsible for high sensitivity, high dynamic range, and strong amplification of chemotaxis signaling. Cryo-electron tomography (cryo-ET) has emerged as a unique tool to visualize bacterial chemotaxis arrays at molecular level. Here we describe a detailed cryo-ET and subtomogram averaging procedure to determine in situ structure of the chemoreceptor arrays in Salmonella minicells. The procedure should be readily applicable to visualize other large macromolecular assemblies in their cellular context.

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.

Mimicking the green fluorescent protein (GFP), multicolor fluorescent polymers possessing enhanced fluorescence have been developed and applied to single-excitation cell imaging. The GFP core chromophore was covalently linked to the azide-functionalized amphiphilic block polymer poly(ethylene glycol)–azide–poly(methyl methacrylate). Through macromolecular assembly into micelles, the fluorescence enhanced and further increased with the elongation of poly(methyl methacrylate) chain due to the segmentation effect of the polymeric framework, which could reduce strong π–π interaction and suppress the chromophore’s conformational motion. By a combination of chemically tailoring the core chromophore and macromolecular assembly strategy, multicolor fluorescent polymers showing a color palette from blue to orange were achieved under similar excitation conditions with the highest emission quantum yield approaching 8%, which is more than 80-fold larger than that of the core chromophore. Moreover, fluorescent emissio...

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. Proteins 2015; 83:1887–1899. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1[CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

How does central nervous system process information? Current theories are based on two tenets: (a) information is transmitted by action potentials, the language by which neurons communicate with each other—and (b) homogeneous neuronal assemblies of cortical circuits operate on these neuronal messages where the operations are characterized by the intrinsic connectivity among neuronal populations. In this view, the size and time course of any spike is stereotypic and the information is restricted to the temporal sequence of the spikes; namely, the “neural code”. However, an increasing amount of novel data point towards an alternative hypothesis: (a) the role of neural code in information processing is overemphasized. Instead of simply passing messages, action potentials play a role in dynamic coordination at multiple spatial and temporal scales, establishing network interactions across several levels of a hierarchical modular architecture, modulating and regulating the propagation of neuronal messages. (b) Information is processed at all levels of neuronal infrastructure from macromolecules to population dynamics. For example, intra-neuronal (changes in protein conformation, concentration and synthesis) and extra-neuronal factors (extracellular proteolysis, substrate patterning, myelin plasticity, microbes, metabolic status) can have a profound effect on neuronal computations. This means molecular message passing may have cognitive connotations. This essay introduces the concept of “supramolecular chemistry”, involving the storage of information at the molecular level and its retrieval, transfer and processing at the supramolecular level, through transitory non-covalent molecular processes that are self-organized, self-assembled and dynamic. Finally, we note that the cortex comprises extremely heterogeneous cells, with distinct regional variations, macromolecular assembly, receptor repertoire and intrinsic microcircuitry. This suggests that every neuron (or group of neurons) embodies different molecular information that hands an operational effect on neuronal computation.

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.

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 β-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.

Protein-protein interactions are essential for the basic biological machinery of the cell. This is important for processes like protein synthesis, enzyme kinetics, molecular assembly and signal transduction. A high number of macromolecular structural complexes are known due to recent advances in structure determination techniques. Therefore, it is of interest to develop an interactive tool to objectively analyze large protein complexes. Hence, we describe the development and utility of a web enabled application named 'Protein-Protein Interaction in Macro-molecular Assembly' (PIMA) for the analysis of large protein assemblies. The intricate details of physical interactions amongst protein subunits in a large complex are presented as simple user preferred interactive network diagrams.

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of ‘client’ proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of substrate ‘client’ proteins. How the ubiquitous chaperone machinery directs its activities towards a specific set of substrates and whether this selectivity could be targeted for therapeutic intervention is of intense research. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone for the FKBP8-HSP90 chaperone complex during the biosynthesis of axonemal dynein heavy chains required for cilia motility. In the absence of ZMYND10, defects in dynein heavy chains trigger broader dynein motor degradation. We show that FKBP8 inhibition phenocopies dynein motor instability in airway cells, and human disease-causing variants of ZMYND10 disrupt its ability to act as FKBP8-HSP90 co-chaperone. Our study indicates that the motile ciliopathy Primary Ciliary Dyskinesia (PCD) should be considered a cell-type specific protein-misfolding disease and opens the potential for rational drug design that could restore specificity to the ubiquitous chaperone apparatus towards dynein subunits.

Staphylococcus aureus pathogenicity islands (SaPIs), such as SaPI1, exploit specific helper bacteriophages, like 80α, for their high frequency mobilization, a process termed ‘molecular piracy’. SaPI1 redirects the helper’s assembly pathway to form small capsids that can only accommodate the smaller SaPI1 genome, but not a complete phage genome. SaPI1 encodes two proteins, CpmA and CpmB, that are responsible for this size redirection. We have determined the structures of the 80α and SaPI1 procapsids to near-atomic resolution by cryo-electron microscopy, and show that CpmB competes with the 80α scaffolding protein (SP) for a binding site on the capsid protein (CP), and works by altering the angle between capsomers. We probed these interactions genetically and identified second-site suppressors of lethal mutations in SP. Our structures show, for the first time, the detailed interactions between SP and CP in a bacteriophage, providing unique insights into macromolecular assembly processes.

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly., Author summary Most animal cells have antenna-like organelles called “cilia”. These organelles have various important functions both in motility and sensing the environment. Motile cilia are essential for moving cells as well as moving fluids across a surface. The waveform of motile cilia requires large macromolecular motors; these are the ciliary dyneins. These dynein complexes are assembled in the cytoplasm in a pathway called preassembly, and then transported into cilia. Defects in this process cause a heterogeneous human disease called primary ciliary dyskinesia that results, for example, in the disruption of the motility of respiratory tract cilia, sperm and nodal cilia during development. The mechanisms of the preassembly pathway are not fully understood. In this study, we use a mutation in the well-conserved DYX1C1/PF23 gene of the green alga, Chlamydomonas reinhardtii. Loss of a conserved domain (DYX) reveals a failure to assemble most ciliary dyneins. Preassembly of inner arm dyneins is particularly affected. We find that if dynein arms are not assembled, dynein subunits in the cytoplasm are unstable. We suggest that DYX1C1 may play a role as a scaffold for other preassembly factors and the dynein subunits.

The shelterin complex is a macromolecular assembly of proteins that binds to and protects telomeric DNA, which composes the ends of all linear chromosomes. Shelterin proteins prevent chromosome ends from fusing together and from eliciting erroneous induction of DNA damage response pathways. Additionally, shelterin proteins play key roles in regulating the recruitment and activation of telomerase, an enzyme that extends telomeric DNA. In fission yeast, Schizosaccharomyces pombe, interactions between the shelterin proteins Ccq1, Tpz1, and Poz1, are important for regulating telomerase-mediated telomere synthesis, and thus, telomere length homeostasis. Here, we used electron microscopy combined with genetic labeling to define the three-dimensional arrangement of the S. pombe Ccq1-Tpz1-Poz1 (CTP) complex. Cross-linking mass spectrometry was used to identify individual residues that are in proximity to the protein-protein interfaces of the assembled CTP complex. Together, our data provide a first glimpse into the architectural design of the CTP complex and reveals unique interactions that are important in maintaining the S. pombe telomere in a non-extendible state.

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide-MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt's lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

Supramolecular polymerization or assembly of proteins or large macromolecular units by a homogeneous nucleation mechanism can be quite slow and require specific solution conditions. In nature, protein assembly is often regulated by molecules that modulate the electrostatic interactions of the protein subunits for various association strengths. The key to this regulation is the coupling of the assembly process with a reversible or irreversible chemical reaction that occurs within the constituent subunits. However, realizing this complex process by the rational design of synthetic molecules or macromolecules remains a challenge. Herein, we use a synthetic polypeptide-grafted comb macromolecule to demonstrate how the in situ modulation of interactions between the charged macromolecules affects their resulting supramolecular structures. The kinetics of structural formation was studied and can be described by a generalized model of nucleated polymerization containing secondary pathways. Basic thermodynamic analysis indicated the delicate role of the electrostatic interactions between the charged subunits in the reaction-induced assembly process. This approach may be applicable for assembling a variety of ionic soft matters that are amenable to chemical reactions in situ.

Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.

Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. The mechanisms underlying this remain key issues. Drosophila embryos provide a superb model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (mammalian Afadin’s homolog) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junctions positioning. Here we define mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane-localization, recruitment to nascent junctions and macromolecular assembly into cables at tricellular junctions. Our data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy/PDZ-GEF is critical for Canoe tricellular junction enrichment but not apical retention. Canoe’s Rap1-interacting RA-domains mediate adherens junction and tricellular junction recruitment but are dispensable for membrane-localization. Our data also support a role for Canoe-multimerization. These multifactorial inputs all shape Canoe localization, correct Bazooka/Par3 and adherens junction positioning, and thus apical-basal polarity. We integrate existing data into a new polarity establishment model.Abbreviations usedα-cat, alpha-catenin; β-cat, beta-catenin; AJ, adherens junction; Arm, Armadillo; Baz, Bazooka; CA, constitutively active; Cno, Canoe; DE-cad,DrosophilaE-cadherin; Dzy, Dizzy; GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GTP, guanosine triphosphate; IF, immunofluorescence; MIP, maximum intensity projection; RA, Ras-associated; RFP, red fluorescent protein; SAJ, spot adherens junction; shRNA, short hairpin RNA; TCJ, tricellular junction; WT, wildtype

Solution processable semiconducting polymers have been under intense investigations due to their diverse applications from printed electronics to biomedical devices. However, controlling the macromolecular assembly across length scales during solution coating remains a key challenge, largely due to the disparity in timescales of polymer assembly and high-throughput printing/coating. Herein we propose the concept of dynamic templating to expedite polymer nucleation and the ensuing assembly process, inspired by biomineralization templates capable of surface reconfiguration. Molecular dynamic simulations reveal that surface reconfigurability is key to promoting template–polymer interactions, thereby lowering polymer nucleation barrier. Employing ionic-liquid-based dynamic template during meniscus-guided coating results in highly aligned, highly crystalline donor–acceptor polymer thin films over large area (>1 cm2) and promoted charge transport along both the polymer backbone and the π–π stacking direction in field-effect transistors. We further demonstrate that the charge transport anisotropy can be reversed by tuning the degree of polymer backbone alignment., Controlling macromolecular assembly across length scales during solution coating of semiconducting polymers is still a challenge. Here the authors show a biomineralization inspired dynamic templating method that expedites polymer nucleation and assembly which allows for surface reconfiguration.

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins β-lactoglobulin (β-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with β-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of β-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio

Bacterial ribosome biogenesis has been an active area of research for more than 30 years and has served as a test-bed for the development of new biochemical, biophysical and structural techniques to understand macromolecular assembly generally. Recent work inspecting the process in vivo has advanced our understanding of the role of ribosome biogenesis factors, the co-transcriptional nature of assembly, the kinetics of the process under sub-optimal conditions, and the rRNA folding and ribosome protein binding pathways. Additionally, new structural work enabled by single-particle electron microscopy has helped to connect in vitro ribosomal protein binding maps to the underlying RNA. This review summarizes the state of these in vivo studies, provides a kinetic model for ribosome assembly under sub-optimal conditions, and describes a framework to compare newly emerging assembly intermediate structures. This article is part of the themed issue ‘Perspectives on the ribosome’.

Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.

Nucleic acids symposium series 45(3), 1501 - 1515(2017). doi:10.1093/nar/gkw1183, The intrinsically disordered p15$^{PAF}$ regulates DNA replication and repair when interacting with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. As many interactions between disordered proteins and globular partners involved in signaling and regulation, the complex between p15$^{PAF}$ and trimeric PCNA is of low affinity, forming a transient complex that is difficult to characterize at a structural level due to its inherent polydispersity. We have determined the structure, conformational fluctuations, and relative population of the five species that coexist in solution by combining small-angle X-ray scattering (SAXS) with molecular modelling. By using explicit ensemble descriptions for the individual species, built using integrative approaches and molecular dynamics (MD) simulations, we collectively interpreted multiple SAXS profiles as population-weighted thermodynamic mixtures. The analysis demonstrates that the N-terminus of p15$^{PAF}$ penetrates the PCNA ring and emerges on the back face. This observation substantiates the role of p15$^{PAF}$ as a drag regulating PCNA processivity during DNA repair. Our study reveals the power of ensemble-based approaches to decode structural, dynamic, and thermodynamic information from SAXS data. This strategy paves the way for deciphering the structural bases of flexible, transient and multivalent macromolecular assemblies involved in pivotal biological processes., Published by Oxford Univ. Press8619, Oxford

The development of an HIV-1/AIDS vaccine has posed great challenges over the last two decades to the international scientific community. The synthesis of a viable vaccine component has gained momentum in recent years after several learning stories with partial success. The moderate efficacy showed by RV144 (ENV-gp120, Gag and Pro) vaccine (Thai trial vaccine) in clinical trials provided impetus to the study during the last decade. The technological challenge in the production of the viral spike as a viable vaccine component is realized since the conclusion of the Thai trial (2009). The results from the NIAID-sponsored South African HVTN 702 (ALVAC-HIV + subtype C gp120/MF59 2700) trial are awaited. The HIV-1 envelope spike [ENV GP160 trimer complex] is being exploited as a promising vaccine candidate in recent years. A number of knowledge points on the viral spike are now available using data derived from several disciplines of biotechnology. The production of a native conformation of the HIV-1 ENV GP160 trimer SPIKE with appropriate glycosylation taking into consideration about 39,000 variants of known GP160 sequences among known subtypes (as available at the LANL database) is found to be nontrivial. The known HIV-1 ENV GP160 trimer SPIKE electron microscopy structure at 4.19 A resolution shows nine interfaces with three different types. Their characteristic features are interesting towards the development of a stable yet effective trimer complex. The GP41-GP41 and GP41-GP120 interfaces are characteristics of equal polar to non-polar residues, and the GP120-GP120 interfaces are predominantly polar in nature. These data find application in the development of an immunologically viable vaccine component.

Lipids are small hydrophobic or amphipathic molecules that may entirely or in part originate by carbanion-based condensations of thioesters and/or by carbocation-based condensations of isoprene units. Further lipids are classified along with prominent examples. The concept of lipids, their molecular shape, macromolecular assembly and packing or shape parameter—surface area, hydrophobic volume and tail length is explained. Kinetically frozen micelles, inverted micelles, and supermicelles along with bilayers—has been explained. This explanation is supported by key features of formation and application aspect as well. Liposomes, vesicles, and GUV have been explained in detail along with related application in the different sectors. The aspect of visualization of various structures using typical visualization tools has been considered. The main focus revolves around X-ray, NMR, and microscopy both confocal and electron microscopy. A section on molecular dynamic simulations, to discuss about computational approach in better understanding, is also considered. The membrane phases, their transitions, and relative curvatures have been dealt in detail. The final section involves study of the role of membrane lipids in cellular process and human health. The aspect of immunotherapy and prospects in the future are also incorporated in the end.

Despite the existence of numerous useful conventions in structural crystallography, for example for the choice of the asymmetric part of the unit cell or of reciprocal space, surprisingly no standards are in use for the placement of the molecular model in the unit cell, often leading to inconsistencies or confusion. A conceptual solution for this problem has been proposed for macromolecular crystal structures based on the idea of the anti-Cheshire unit cell. Here, a program and server (calledACHESYM; http://achesym.ibch.poznan.pl) are presented for the practical implementation of this concept. In addition, the first task ofACHESYMis to find an optimal (compact) macromolecular assembly if more than one polymer chain exists.ACHESYMprocesses PDB (atomic parameters andTLSmatrices) and mmCIF (diffraction data) input files to produce a new coordinate set and to reindex the reflections and modify their phases, if necessary.

Macromolecular assembly at the interface between water and a hydrophobic surface underlies some of the most important biological and environmental processes on the planet. Important model systems for understanding this assembly at such interfaces are polyelectrolytes that have hydrophilic units interspersed along a hydrophobic polymer backbone, similar to many biological macromolecules. This feature article provides recent advances in the molecular factors that contribute to the adsorption and assembly of carboxylic acid-containing polyelectrolytes at the carbon tetrachloride–water interface, a model system for an oil–water interface. Using vibrational sum frequency spectroscopy and interfacial tension techniques, these unique set of studies presented herein identify the factors that dictate whether or not polyelectrolytes will adsorb to the oil–water interface and also describe the specifics of the adsorption process that depend upon factors such as polymer size, charge density, hydrophobicity, conformat...

Periderms present in plant barks are essential protective barriers to water diffusion, mechanical breakdown, and pathogenic invasion. They consist of densely packed layers of dead cells with cell walls that are embedded with suberin. Understanding the interplay of molecular structure, dynamics, and biomechanics in these cell wall-associated insoluble amorphous polymeric assemblies presents substantial investigative challenges. We report solid-state NMR coordinated with FT-IR and tensile strength measurements for periderms from native and wound-healing potatoes and from potatoes with genetically modified suberins. The analyses include the intact suberin aromatic–aliphatic polymer and cell-wall polysaccharides, previously reported soluble depolymerized transmethylation products, and undegraded residues including suberan. Wound-healing suberized potato cell walls, which are 2 orders of magnitude more permeable to water than native periderms, display a strikingly enhanced hydrophilic–hydrophobic balance, a degradation-resistant aromatic domain, and flexibility suggestive of an altered supramolecular organization in the periderm. Suppression of ferulate ester formation in suberin and associated wax remodels the periderm with more flexible aliphatic chains and abundant aromatic constituents that can resist transesterification, attenuates cooperative hydroxyfatty acid motions, and produces a mechanically compromised and highly water-permeable periderm.