Dynamic Macromolecular Complexes Research Articles

Promyelocytic Leukemia Protein Potently Restricts Human Cytomegalovirus Infection in Endothelial Cells.

PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate–early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells.

Differential Paralog-Specific Expression of Multiple Small Subunit Proteins Cause Variations in Rpl42/eL42 Incorporation in Ribosome in Fission Yeast

Ribosomes within a cell are commonly viewed as biochemically homogenous RNA–protein super-complexes performing identical functions of protein synthesis. However, recent evidence suggests that ribosomes may be a more dynamic macromolecular complex with specialized roles. Here, we present extensive genetic and molecular evidence in the fission yeast S. pombe that the paralogous genes for many ribosomal proteins (RPs) are functionally different, despite that they encode the same ribosomal component, often with only subtle differences in the sequences. Focusing on the rps8 paralog gene deletions rps801d and rps802d, we showed that the mutant cells differ in the level of Rpl42p in actively translating ribosomes and that their phenotypic differences reside in the Rpl42p level variation instead of the subtle protein sequence difference between Rps801p and Rps802p. Additional 40S ribosomal protein paralog pairs also exhibit similar phenotypic differences via differential Rpl42p levels in actively translating ribosomes. Together, our work identifies variations in the Rpl42p level as a potential form of ribosome heterogeneity in biochemical compositions and suggests a possible connection between large and small subunits during ribosome biogenesis that may cause such heterogeneity. Additionally, it illustrates the complexity of the underlying mechanisms for the genetic specificity of ribosome paralogs.

An Evaluation of the Stability of Splicing Protein Dib1 Through Circular Dichroism Spectroscopy

Pre‐messenger RNA (pre‐mRNA) splicing is the process in which introns, noncoding regions of DNA, are removed and exons, coding regions of DNA, are ligated to produce a mature mRNA transcript. This process is mediated by the spliceosome, a dynamic macromolecular complex composed of five small nuclear ribonucleoproteins and other protein factors. At the catalytic center of the splicing machinery is the evolutionarily conserved yeast protein, Dib1, composed of 143 amino acids essential for splicing and cell viability. Dib1 and its human ortholog, hDim1, have demonstrated peptidase activity, specifically autocleavage of the last thirteen and fourteen amino acids, respectively, of their carboxy‐terminal tails. The mechanism and function of this autocleavage activity of the carboxy‐terminal tail is uncharacterized.Previous yeast growth assays comparing mutants to wildtype Dib1 have established that deletion of fifteen and sixteen amino acids off the carboxy‐terminal tail results in temperature‐sensitivity of yeast cell growth at 37ºC. Since Dib1 and splicing are essential for cell viability, the temperature‐sensitive mutants suggest that truncations to the carboxy‐terminal tail of Dib1 may result in defective splicing. Thus, the carboxy‐terminal tail may play a direct role in splicing or destabilize splicing complexes. To address these two possible hypotheses, we are investigating the effects of the carboxy‐terminal truncations on Dib1 stability. Protein purification of wild type and a series of truncated Dib1 mutants were performed. Circular dichroism spectroscopy employing increasing concentrations of guanidinium hydrochloride, a protein denaturant, was used to determine the stability of Dib1 and its truncated mutants. Overall, our findings show that Dib1 stability is directly linked to the length of the tail region and lead us to speculate that the tail is critical for stabilization of the spliceosomal complex.

Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to get a comprehensive picture of cellular behavior and to understand biological systems. In the past two decades, affinity purification coupled to mass spectrometry has become a powerful tool to comprehensively study interaction networks and their assemblies. To overcome initial limitations of the approach, in particular, the effect of protein and RNA degradation, loss of transient interactors, and poor overall yield of intact complexes from cell lysates, various modifications to affinity purification protocols have been devised over the years. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol as described here has been optimized for the yeast S. cerevisiae.

HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes.

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).

Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a 'closed' and an 'open' conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution.

The Role of the U5 snRNP in Genetic Disorders and Cancer.

Pre-mRNA splicing is performed by the spliceosome, a dynamic macromolecular complex consisting of five small uridine-rich ribonucleoprotein complexes (the U1, U2, U4, U5, and U6 snRNPs) and numerous auxiliary splicing factors. A plethora of human disorders are caused by genetic variants affecting the function and/or expression of splicing factors, including the core snRNP proteins. Variants in the genes encoding proteins of the U5 snRNP cause two distinct and tissue-specific human disease phenotypes – variants in PRPF6, PRPF8, and SNRP200 are associated with retinitis pigmentosa (RP), while variants in EFTUD2 and TXNL4A cause the craniofacial disorders mandibulofacial dysostosis Guion-Almeida type (MFDGA) and Burn-McKeown syndrome (BMKS), respectively. Furthermore, recurrent somatic mutations or changes in the expression levels of a number of U5 snRNP proteins (PRPF6, PRPF8, EFTUD2, DDX23, and SNRNP40) have been associated with human cancers. How and why variants in ubiquitously expressed spliceosome proteins required for pre-mRNA splicing in all human cells result in tissue-restricted disease phenotypes is not clear. Additionally, why variants in different, yet interacting, proteins making up the same core spliceosome snRNP result in completely distinct disease outcomes – RP, craniofacial defects or cancer – is unclear. In this review, we define the roles of different U5 snRNP proteins in RP, craniofacial disorders and cancer, including how disease-associated genetic variants affect pre-mRNA splicing and the proposed disease mechanisms. We then propose potential hypotheses for how U5 snRNP variants cause tissue specificity resulting in the restricted and distinct human disorders.

Scribble co-operatively binds multiple \u03b11D-adrenergic receptor C-terminal PDZ ligands

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) – key regulators of cardiovascular and central nervous system function – form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

Co-regulated gene expression of splicing factors as drivers of cancer progression

Splicing factors (SFs) act in dynamic macromolecular complexes to modulate RNA processing. To understand the complex role of SFs in cancer progression, we performed a systemic analysis of the co-regulation of SFs using primary tumor RNA sequencing data. Co-regulated SFs were associated with aggressive breast cancer phenotypes and enhanced metastasis formation, resulting in the classification of Enhancer- (21 genes) and Suppressor-SFs (64 genes). High Enhancer-SF levels were related to distinct splicing patterns and expression of known oncogenic pathways such as respiratory electron transport, DNA damage and cell cycle regulation. Importantly, largely identical SF co-regulation was observed in almost all major cancer types, including lung, pancreas and prostate cancer. In conclusion, we identified cancer-associated co-regulated expression of SFs that are associated with aggressive phenotypes. This study increases the global understanding of the role of the spliceosome in cancer progression and also contributes to the development of strategies to cure cancer patients.

Energetics and Interfacial Interactions of Spliceosomal Protein Dib1 Predicted with MD Simulations

The spliceosome is the highly dynamic macromolecular complex responsible for catalyzing the excision of introns from pre‐messenger RNA. The spliceosome consists of five small nuclear ribonucleoproteins as well as several additional proteins. As part of a complex assembly pathway, the essential protein Dib1 must leave the spliceosome. Experimental analysis has found a temperature sensitive phenotype of Dib1 (TS‐Dib1, F85A) that hinders splicing activity. This phenotype has also been hypothesized to perturb spliceosome assembly. Our current work uses atomistic molecular dynamic (MD) simulations to study the molecular interactions in the region of the spliceosome around Dib1 in order to determine what energetic and structural changes result from this point mutation.Starting with recently published cryo‐EM structures, the spliceosome was reduced to only include components in close proximity to Dib1 (either native or F85A). The reduced Dib1 structure consists of three snRNAs (U4, U5, and U6) and four proteins (Prp6, Prp8, Brr2, Prp31). Both systems, containing either native Dib1 or mutant Dib1, were simulated using atomistic MD simulations to identify energetic differences between native Dib1 and mutant TS‐Dib1. The simulation conditions mimicked the biological conditions of previous bulk splicing assays, with an aqueous solvent, 0.1 M salt, and temperature differences that have experimentally shown TS‐Dib1 inactivation. The MD simulations revealed a lower global binding energy in the TS‐Dib1 system compared to the native Dib1 system. Additionally, interfacial hydrogen bonding patterns displayed increased hydrogen bonding among Prp6, Prp8, and Dib1 in the native Dib1 system that were not present in the TS‐Dib1 system, which could contribute to the lower global binding energy. The results from the MD simulations support the experimentally observed temperature sensitivity in the TS‐Dib1 system and could help explain the role of Dib1 in spliceosome assembly.Support or Funding InformationNIH R15GM120720, Robert A Welch Foundation Grant W‐1905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein-Cross-Linking zur Aufkl\xe4rung von komplexen Strukturen

Cryo-electron microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes. To characterize less well defined regions in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the arrangement of domains and amino acids. CX-MS involves covalent linkage of protein residues close to each other and identifying these connections by mass spectrometry. Here, we summarise the advances of CX-MS and its integration with cryo-EM for structural reconstruction.

VPS28, an ESCRT-I protein, regulates mitotic spindle organization via Gβγ, EG5 and TPX2

The mitotic spindle is a dynamic macromolecular complex essential for chromosome segregation. ESCRT (endosomal sorting complexes required for transport) proteins are emerging as relevant mitotic players putatively recruiting spindle organizers. Here, we describe that VPS28, an ESCRT-I component, directly interacts with Gβγ, a signaling heterodimer with documented impact on microtubule dynamics, composing a novel organizer of the mitotic spindle aster. We found that VPS28 localizes to mitotic microtubules where it recruits Gβγ. Reducing VPS28 expression impairs kinesin Eg5 and TPX2 localization to the mitotic spindle. The interaction between VPS28 and Gβγ involves the carboxyl-terminal region of VPS28, which usually interacts with VPS36, its regular partner at multivesicular bodies. The VPS28-Gβγ complex is better constituted in the presence of Gα independently of G protein coupled receptor-stimulation, suggesting an intrinsic mechanism of regulation by which this novel complex contributes to mitotic spindle organization in mammalian cells.

Complementary Use of Electron Cryomicroscopy and X-Ray Crystallography: Structural Studies of Actin and Actomyosin Filaments.

Visualization of macromolecular structures is essential for understanding the mechanisms of biological functions because they are all determined by the structure and dynamics of macromolecular complexes. Electron cryomicroscopy (cryoEM) and image analysis has become a powerful tool for structural studies because of recent technical developments in microscope optics, cryostage control, image detection and the methods of sample preparation. In particular, the recent development of CMOS-based direct electron detectors with high sensitivity, high resolution and high frame rate has revolutionized the field of structural biology by making near-atomic resolution structural analysis possible from small amounts of solution samples. However, for some biological systems, it is still difficult to reach high resolution due to somewhat flexible nature of the structure, and a complementary use of cryoEM with X-ray crystallography is essential and useful to gain mechanistic understanding of the biological functions and mechanisms. We will describe our strategy for the structural analyses of actin filament and actomyosin rigor complex and the biological insights we gained from these structures.

Combining X-ray and Cryo-EM to study large and dynamic macromolecular complexes

Combining X-ray and Cryo-EM to study large and dynamic macromolecular complexes

Methods for Preparing Cryo-EM Grids of Large Macromolecular Complexes.

The recent resolution revolution in cryo-electron microscopy has generated a huge interest in the technique for determining atomic resolution structures of large and dynamic macromolecular complexes that are intractable to crystallography and NMR. A key element of success in cryo-EM is the quality of the specimen vitrified on the cryo-EM grid. In this chapter we outline methods for cryo-EM grid sample preparation.

Structure and Conformational Dynamics of the Human Spliceosomal Bact Complex

The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human Bact spliceosome at 3.4Å resolution. In the Bact state, the spliceosome is activated but not catalytically primed, so that it is functionally blocked prior to the first catalytic step of splicing. The spliceosomal core is similar to the yeast Bact spliceosome; important differences include the presence of the RNA helicase aquarius and peptidyl prolyl isomerases. To examine the overall dynamic behavior of the purified spliceosome, we developed a principal component analysis-based approach. Calculating the energy landscape revealed eight major conformational states, which we refined to higher resolution. Conformational differences of the highly flexible structural components between these eight states reveal how spliceosomal components contribute to the assembly of the spliceosome, allowing it to generate a dynamic interaction network required for its subsequent catalytic activation.

Structures of Dynamic Protein Complexes: Hybrid Techniques to Study MAP Kinase Complexes and the ESCRT System.

The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

A recurring challenge in cell biology is to define themolecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.

Preparation of DNA Substrates and Functionalized Glass Surfaces for Correlative Nanomanipulation and Colocalization (NanoCOSM) of Single Molecules.

Simultaneous nanomanipulation and colocalization of single molecules (NanoCOSM) provides a unique opportunity to correlate the mechanical properties and activities of biomolecules with their conformational states or states of assembly as part of dynamic macromolecular complexes. This opens the door to real-time single-molecule analysis of the correlations between structure, function, and composition of large multicomponent protein complexes.

Coarse-Graining of Volumes for Modeling of Structure and Dynamics in Electron Microscopy: Algorithm to Automatically Control Accuracy of Approximation

Coarse-graining (or granularization) of structures from transmission electron microscopy (EM volumes) has been shown to be useful for a variety of structural analysis applications. Several methods perform coarse-graining of EM volumes using hard spheres or 3D Gaussian functions but they do not allow controlling automatically the volume approximation accuracy. To tackle this problem, we recently developed such a method. It is currently used by 3DEM Loupe web server and HEMNMA software to study macromolecular dynamics based on coarse-grained representations of EM volumes. In this paper, we give a detailed description of the implemented algorithm and fully analyze its performance, which was out of scope of our previous papers. The performance is analyzed in a controlled environment, in the context of studying structure and dynamics of macromolecular complexes. We show that this technique allows computing structures that are similar to atomic structures, by analyzing intermediate-resolution volumes. Additionally, we show that it allows sharpening of intermediate-resolution volumes. The full algorithm description allows its implementation in any other software package.