[Assembly and Disassembly of the Nuclear Pore Complex: A View from the Structural Side].

Nucleocytoplasmic exchange in the cell occurs through the nuclear pore complexes (NPCs). NPCs are large multiprotein complexes with octagonal symmetry about their axis and imperfect mirror symmetry about a plane parallel with the nuclear envelop (NE). NPC fuses the inner and outer nuclear membranes and opens up a channel between nucleus and cytoplasm. NPC is built of nucleoporins. Each nucleoporin occurs in at least eight copies per NPC. Inside the NPC a permeability barrier forms by which NPCs can provide fast and selectable transport of molecules from one side of the nuclear membrane to the other. NPC architecture is based on hierarchical principle of organization. Nucleoporins are integrated into complexes that oligomerizes into bigger octomeric high-order structures. These structures are the main components of NPCs. In the first part of this work, the main attention is paid to NPC structure and nucleoporin properties. The second part is dedicated to mechanisms of NPC assembly and disassembly at different stages of the cell cycle.

Synthetic Antibodies Detect Distinct Cellular States of Chromosome Passenger Complex Proteins.

High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.

Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes.

In mitochondrial oxidative phosphorylation (Ox-Phos), individual electron transport chain complexes are thought to assemble into supramolecular entities termed supercomplexes (SCs). The technique of blue native (BN) gel electrophoresis has emerged as the method of choice for analyzing SCs. However, the process of sample extraction for BN gel analysis is somewhat tedious and introduces the possibility for experimental artifacts. Here we outline a streamlined method that eliminates a centrifugation step and provides a more representative sampling of a population of mitochondria on the final gel. Using this method, we show that SC composition does not appear to change dynamically with altered mitochondrial function.

Strategy for high-throughput identification of protein complexes by array-based multi-dimensional liquid chromatography-mass spectrometry.

Comprehensive elucidation of the composition of multiprotein complexes in model organisms is essential to understand conserved biological systems, but large-scale mapping physical association networks is still challenging due to limited throughput of present methods. In this work, a strategy coupling array-based online two-dimensional liquid chromatography (array-based 2D-LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was demonstrated for high throughput and in-depth identification of protein complexes from cultured human HeLa cell extracts. Mixed-bed ion-exchange column was employed as the first dimensional (1stD) separating mode and an array consisting of eight reversed phase columns was developed as the second dimensional (2ndD) mode. Taking advantage of array parallel strategy, this online system showed an 8-fold increase in throughput. After array-based online 2D-LC separation, altogether 256 × 2ndD fractions were collected for further LC-MS/MS analysis. Public databases of protein-protein interaction (PPI) and co-elution curves identified by LC-MS were applied to reconstruct the protein complexes. A rigorous inspection was operated by cataloging the protein complexes into chromatographic fractions to minimize the number of false positives. As result, a total number of 4,436 proteins were identified and 26,092 elution curves were graphed. A network consisting of 47,745 PPIs was established among 2,201 proteins and presented 1,530 putative protein complexes with high confidence. Most of the identified PPIs were linked to diverse biological processes and may reveal further disease mechanism and therapeutic strategy.

Ion Imaging of Native Protein Complexes Using Orthogonal Time-of-Flight Mass Spectrometry and a Timepix Detector.

Native mass spectrometry (native MS) has emerged as a powerful technique to study the structure and stoichiometry of large protein complexes. Traditionally, native MS has been performed on modified time-of-flight (TOF) systems combined with detectors that do not provide information on the arrival coordinates of each ion at the detector. In this study, we describe the implementation of a Timepix (TPX) pixelated detector on a modified orthogonal TOF (O-TOF) mass spectrometer for the analysis and imaging of native protein complexes. In this unique experimental setup, we have used the impact positions of the ions at the detector to visualize the effects of various ion optical parameters on the flight path of ions. We also demonstrate the ability to unambiguously detect and image individual ion events, providing the first report of single-ion imaging of protein complexes in native MS. Furthermore, the simultaneous space- and time-sensitive nature of the TPX detector was critical in the identification of the origin of an unexpected TOF signal. A signal that could easily be mistaken as a fragment of the protein complex was explicitly identified as a secondary electron signal arising from ion-surface collisions inside the TOF housing. This work significantly extends the mass range previously detected with the TPX and exemplifies the value of simultaneous space- and time-resolved detection in the study of ion optical processes and ion trajectories in TOF mass spectrometers.

Comparison of loop extrusion and diffusion capture as mitotic chromosome formation pathways in fission yeast.

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long Schizosaccharomyces pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin compaction. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

Solid-state NMR approaches to investigate large enzymes in complex with substrates and inhibitors.

Enzyme catalysis is omnipresent in the cell. The mechanisms by which highly evolved protein folds enable rapid and specific chemical transformation of substrates belong to the marvels of structural biology. Targeting of enzymes with inhibitors has immediate application in drug discovery, from chemotherapeutics over antibiotics to antivirals. NMR spectroscopy combines multiple assets for the investigation of enzyme function. The non-invasive technique can probe enzyme structure and dynamics and map interactions with substrates, cofactors and inhibitors at the atomic level. With experiments performed at close to native conditions, catalytic transformations can be monitored in real time, giving access to kinetic parameters. The power of NMR in the solid state, in contrast with solution, lies in the absence of fundamental size limitations, which is crucial for enzymes that are either membrane-embedded or assemble into large soluble complexes exceeding hundreds of kilodaltons in molecular weight. Here we review recent progress in solid-state NMR methodology, which has taken big leaps in the past years due to steady improvements in hardware design, notably magic angle spinning, and connect it to parallel biochemical advances that enable isotope labelling of increasingly complex enzymes. We first discuss general concepts and requirements of the method and then highlight the state-of-the-art in sample preparation, structure determination, dynamics and interaction studies. We focus on examples where solid-state NMR has been instrumental in elucidating enzyme mechanism, alone or in integrative studies.

Hands on Native Mass Spectrometry Analysis of Multi-protein Complexes.

By maintaining intact multi-protein complexes in the gas-phase, native mass spectrometry provides their molecular weight with very good accuracy compared to other methods (typically native PAGE or SEC-MALS) (Marcoux and Robinson, Structure 21:1541-1550, 2013). Besides, heterogeneous samples, in terms of both oligomeric states and ligand-bound species can be fully characterized. Here we thoroughly describe the analysis of several oligomeric protein complexes ranging from a 16 = kDa dimer to a 801-kDa tetradecameric complex on different instrumental setups.

Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis.

By using negatively charged Coomassie brilliant blue G-250 dye to induce a charge shift on proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) allows resolution of enzymatically active multiprotein complexes extracted from cellular or subcellular lysates while retaining their native conformation. BN-PAGE was first developed to analyze the size, composition, and relative abundance of the complexes and supercomplexes that form the mitochondrial respiratory chain and OXPHOS system. Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results. For complete details on the use and execution of this protocol, please refer to Lobo-Jarne et al. (2018) and Timón-Gómez et al. (2020).

High-Pressure Electrospray Ionization Yields Supercharged Protein Complexes from Native Solutions While Preserving Noncovalent Interactions.

Increasing charge state of protein complexes from native solutions while preserving noncovalent interactions in native mass spectrometry (MS) offers great opportunity to gain deeper insights into gas-phase protein structures. Several previous studies have disclosed the possibility of high pressure in supercharging small proteins, whereas its capability to supercharge large protein assemblies under native conditions and how it might affect protein structures remain open questions. Herein, we demonstrated that the high-pressure-induced supercharging strategy affords unique advantages of supercharging protein complexes with the highest charge state surpassing the Rayleigh limit (ZR) and concurrently preserving native-like topology. By examining 32 proteins and protein complexes with molecular weights (MWs) ranging from 8.58 to 801 kDa, we demonstrated that the increased average charge states of macromolecular ions have a strong dependence on the surface areas of native protein conformations and MWs. Factors that might contribute to the high-pressure-induced supercharging capability toward macromolecular ions were discussed. Furthermore, using collision cross section (CCS) variation as a function of charge state, we investigate the effects of gas pressure and charge states on gas-phase structures of proteins and protein complexes. Smaller proteins have the largest CCS variations once supercharged, while macromolecular protein complexes are less affected. The results revealed that both surface density of charge and charged surface basic residues contribute to the observed CCS-charge disciplines for all the macromolecules investigated. Taken together, the results presented here indicate that increasing gas pressure in the ion source affords a rapid, simple, and controlled supercharging method, offering the potency of facilitating further applications of native top-down MS analysis with improved transmission, fragmentation, and detection efficiency.

Native LESA TWIMS-MSI: Spatial, Conformational, and Mass Analysis of Proteins and Protein Complexes.

We have previously demonstrated native liquid extraction surface analysis (LESA) mass spectrometry imaging of small intact proteins in thin tissue sections. We also showed calculation of collision cross sections for specific proteins extracted from discrete locations in tissue by LESA traveling wave ion mobility spectrometry (TWIMS). Here, we demonstrate an integrated native LESA TWIMS mass spectrometry imaging (MSI) workflow, in which ion mobility separation is central to the imaging experiment and which provides spatial, conformational, and mass information on endogenous proteins in a single experiment. The approach was applied to MSI of a thin tissue section of mouse kidney. The results show that the benefits of integration of TWIMS include improved specificity of the ion images and the capacity to calculate collision cross sections for any protein or protein complex detected in any pixel (without a priori knowledge of the presence of the protein).

Direct Mass Spectrometry Analysis of Protein Complexes and Intact Proteins up to >70 kDa from Tissue.

Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.

Complex-centric proteome profiling by SEC-SWATH-MS.

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error-controlled, complex-centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub-complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity.

Global Identification of Protein Complexes within the Membrane Proteome of Arabidopsis Roots Using a SEC-MS Approach.

Biological processes consist of several consecutive and interacting steps as, for example, in signal transduction cascades or metabolic reaction chains. These processes are regulated by protein-protein interactions and the formation of larger protein complexes, which also occur within biological membranes. To gain a large-scale overview of complex-forming proteins and the composition of such complexes within the cellular membranes of Arabidopsis roots, we use the combination of size-exclusion chromatography and mass spectrometry. First, we identified complex-forming proteins by a retention shift analysis relative to expected retention times of monomeric proteins during size-exclusion chromatography. In a second step we predicted complex composition through pairwise correlation of elution profiles. As result we present an interactome of 963 proteins within cellular membranes of Arabidopsis roots. Identification of complex-forming proteins was highly robust between two independently grown root proteomes. The protein complex composition derived from pairwise correlations of coeluting proteins reproducibly identified stable protein complexes (ribosomes, proteasome, mitochondrial respiratory chain supercomplexes) but showed higher variance between replicates regarding transient interactions (e.g., interactions with kinases) within membrane protein complexes.

Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.

Three-Fragment Fluorescence Complementation for Imaging of Ternary Complexes under Physiological Conditions.

Protein-protein interactions (PPIs) occur in a vast variety of cellular processes, and many processes are regulated by multiple protein interactions. Identification of PPIs is essential for the analysis of biological pathways and to further understand underlying molecular mechanisms. However, visualization and identification of multiprotein complexes, including ternary complexes in living cells under physiological conditions, remains challenging. In this work, we reported a three-fragment fluorescence complementation (TFFC) by splitting the Venus fluorescent protein for visualizing ternary complexes in living cells under physiological conditions. With this Venus-based TFFC system, we identified the multi-interaction of weak-affinity ternary complexes under physiological conditions. The TFFC system was further applied to the analysis of multi-interactions during the HIV-1 integration process, revealing the important role of the barrier-to-autointegration factor protein in HIV-1 integration. This TFFC system provides a useful tool for visualizing and identifying ternary complexes in living cells under physiological conditions.

Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.

Assessment of the cellular localisation of the annexin A2/S100A10 complex in human placenta.

The AnxA2/S100A10 complex has been implicated in various placental functions but although the localisation of these proteins individually has been studied, there is no information about the localisation of their complex in situ at the cellular level. Using the proximity ligation technique, we have investigated the in situ localisation of AnxA2/S100A10 complex in the placenta and have compared this with the location patterns of the individual proteins. High levels of expression of AnxA2/S100A10 complexes were observed in the amniotic membrane and in blood vessel endothelial cells. Lower levels were detected in the brush border area of the syncytium and in the trophoblasts. Immunohistochemical analysis of AnxA2 and S100A10 individually revealed broadly similar patterns of localisation. The brush border staining pattern suggests that in this location at least some of the AnxA2 is not in complex with S100A10. The formal location of the AnxA2/S100A10 complex is compatible with a role in cell-cell interaction, intracellular transport and secretory processes and regulation of cell surface proteases, implying contributions to membrane integrity, nutrient exchange, placentation and vascular remodelling in different parts of the placenta. Future applications will allow specific assessment of the association of the complex with pathophysiological disorders.

Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes.

Native mass spectrometry (MS) encompasses methods to keep noncovalent interactions of biomolecular complexes intact in the gas phase throughout the instrument and to measure the mass-to-charge ratios of supramolecular complexes directly in the mass spectrometer. Electrospray ionization (ESI) in nondenaturing conditions is now an established method to characterize noncovalent systems. Matrix-assisted laser desorption/ionization (MALDI), on the other hand, consumes low quantities of samples and largely tolerates contaminants, making it a priori attractive for native MS. However, so-called native MALDI approaches have so far been based on solid deposits, where the rapid transition of the sample through a solid state can engender the loss of native conformations. Here we present a new method for native MS based on liquid deposits and MALDI ionization, unambiguously detecting intact noncovalent protein complexes by direct desorption from a liquid spot for the first time. To control for aggregation, we worked with HUαß, a heterodimer that does not spontaneously rearrange into homodimers in solution. Screening through numerous matrix solutions to observe first the monomeric protein, then the dimer complex, we settled on a nondenaturing binary matrix solution composed of acidic and basic organic matrices in glycerol, which is stable in vacuo. The role of temporal and spatial laser irradiation patterns was found to be critical. Both a protein-protein and a protein-ligand complex could be observed free of aggregation. To minimize gas-phase dissociation, source parameters were optimized to achieve a conservation of complexes above 50% for both systems. Graphical Abstract ᅟ.

Cdk phosphorylation licenses Kif4A chromosome localization required for early mitotic progression.

The chromokinesin Kif4A controls proper chromosome condensation, congression/alignment, and cytokinesis to ensure faithful genetic inheritance. Here, we report that Cdk phosphorylation of human Kif4A at T1161 licenses Kif4A chromosomal localization, which, in turn, controls Kif4A early mitotic function. Phosphorylated Kif4A (Kif4AWT) or Cdk phospho-mimetic Kif4A mutant (Kif4ATE) associated with chromosomes and condensin I (non-SMC subunit CAP-G and core subunit SMC2) to regulate chromosome condensation, spindle morphology, and chromosome congression/alignment in early mitosis. In contrast, Cdk non-phosphorylatable Kif4A mutant (Kif4ATA) could neither localize on chromosomes nor associate with CAP-G and SMC2. Furthermore, Kif4ATA could not rescue defective chromosome condensation, spindle morphology, or chromosome congression/alignment in cells depleted of endogenous Kif4A, which activated a mitotic checkpoint and delayed early mitotic progression. However, targeting Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1 resulted in restoration of chromosome and spindle functions of Kif4A, similar to Kif4AWT and Kif4ATE, in cells depleted of endogenous Kif4A. Thus, our results demonstrate that Cdk phosphorylation-licensed chromosomal localization of Kif4A plays a critical role in regulating early mitotic functions of Kif4A that are important for early mitotic progression.