[Show abstract][Hide abstract] ABSTRACT: At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ɛ is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α-primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ɛ, first threading through the Mcm2-7 ring and then making a U-turn at the bottom and reaching Pol ɛ at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein coupled receptors, and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of 5 domains, binds and inhibits the enzymatic activity of Class IA PI3Ks. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation (AUC) studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide with unique electrostatic character. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy (FFS). Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle X-ray scattering (SAXS) and chemical cross-linking structural analysis of the discrete dimer. These experimental data are used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.
Journal of Biological Chemistry 10/2015; DOI:10.1074/jbc.M115.689604 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.
[Show abstract][Hide abstract] ABSTRACT: DNA double-strand break repair involves phosphorylation of histone variant H2AX ('γH2AX'), which accumulates in foci at sites of DNA damage. In current models, the recruitment of multiple DNA repair proteins to γH2AX foci depends mainly on recognition of this 'mark' by a single protein, MDC1. However, DNA repair proteins accumulate at γH2AX sites without MDC1, suggesting that other 'readers' of this mark exist. Here, we use a quantitative chemical proteomics approach to profile direct, phospho-selective γH2AX binders in native proteomes. We identify γH2AX binders, including the DNA repair mediator 53BP1, which we show recognizes γH2AX through its BRCT domains. Furthermore, we investigate the targeting of wild-type 53BP1, or a mutant form deficient in γH2AX binding, to chromosomal breaks resulting from endogenous and exogenous DNA damage. Our results show how direct recognition of γH2AX modulates protein localization at DNA damage sites, and suggest how specific chromatin mark-reader interactions contribute to essential mechanisms ensuring genome stability.
Nature Chemical Biology 09/2015; 11(10). DOI:10.1038/nchembio.1908 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screening method that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles for even well-studied proteins. Our approach is robust, economical and automatable, providing inroads to the rigorous, systematic dissection of cellular interactomes.
[Show abstract][Hide abstract] ABSTRACT: Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls.
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that the efficiency of ion transmission from atmosphere to vacuum through stainless steel electrodes that contain slowly divergent conical duct (ConDuct) channels can be close to 100%. Here, we explore the properties of 2.5-cm-long electrodes with angles of divergence of 0°, 1°, 2°, 3°, 5°, 8°, 13°, and 21°, respectively. The ion transmission efficiency was observed to jump from 10-20% for the 0° (straight) channels to 90-95% for channels with an angle of divergence as small as 1°. Furthermore, the 2-3° ConDuct electrodes produced extraordinarily low divergence ion beams that propagated in a laser-like fashion over long distances in vacuum. To take advantage of these newly discovered properties, we constructed a novel atmosphere-to-vacuum ion interface utilizing a 2° ConDuct as an inlet electrode and compared its ion transmission efficiency with that of the interface used in the commercial (Thermo Fisher Scientific, San Jose, CA, USA) Velos Orbitrap and Q Exactive mass spectrometers. We observed that the ConDuct interface transmitted up to 17 times more ions than the commercial reference interface and also yielded improved signal-to-noise mass spectra of peptides. We infer from these results that the performance of many current atmosphere-to-vacuum interfaces utilizing metal capillaries can be substantially improved by replacing them with 1° or 2° metal ConDuct electrodes, which should preserve the convenience of supplying ion desolvation energy by heating the electrode while greatly increasing the efficiency of ion transmission into the mass spectrometer.
Journal of the American Society for Mass Spectrometry 02/2015; 26(4). DOI:10.1007/s13361-014-1063-0 · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metabotropic glutamate receptor 5 (mGluR5) regulates excitatory postsynaptic signaling in the central nervous system (CNS) and is implicated in various CNS disorders. Protein kinase A (PKA) signaling is known to play a critical role in neuropsychiatric disorders such as Parkinson's disease, schizophrenia and addiction. Dopamine signaling is known to modulate the properties of mGluR5 in a cAMP- and PKA-dependent manner, suggesting that mGluR5 may be a direct target for PKA. Our study identifies mGluR5 at Ser870 as a direct substrate for PKA phosphorylation and demonstrates that this phosphorylation plays a critical role in the PKA-mediated modulation of mGluR5 functions such as extracellular signal-regulated kinase (ERK) phosphorylation and intracellular Ca(2+) oscillations. The identification of the molecular mechanism by which PKA signaling modulates mGluR5-mediated cellular responses contributes to the understanding of the interaction between dopaminergic and glutamatergic neuronal signaling. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Journal of Neurochemistry 02/2015; 132(6). DOI:10.1111/jnc.13038 · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have discovered that an electrode containing a conical channel with a small angular divergence can transmit into the vacuum almost 100% of an electrospray ion current produced at atmospheric pressure. Our first implementation of such a conical duct, which we term "ConDuct," uses a conductive plastic pipette tip containing an approximately 1.6° divergent channel at its entrance. We observed that the beam formed by the ConDuct electrode has a very low divergence (less than 1°) and persists for long distances in vacuum. Intrigued by these properties, we incorporated this electrode into a novel atmosphere-to-vacuum ion transmission interface, and devised a technique for evaluating its performance relative to the commercial reference interfaces that contain heated metal capillaries. We determined that our new interface transmits at least 400 times more ions than the commercial Thermo LCQ DECA XP atmosphere-to-vacuum interface and 2 to 3 times more than the commercial interface in the Thermo Velos Orbitrap and the Q Exactive mass spectrometers. We conclude that it might be possible to optimize the properties of the transmitted ions further by manufacturing ConDuct inlet electrodes from metal rather than conductive plastic and by determining the optimum angle of channel divergence and channel length.
Journal of the American Society for Mass Spectrometry 01/2015; 26(4). DOI:10.1007/s13361-014-1062-1 · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Membrane fouling, the process in which proteins adhere to the external and internal surface of synthetic membranes, is considered a major limitation in membrane filtration of biofluids. This causes permeation flux and changes in selectivity, with the overall effect of reducing membrane performance and increasing process cost. In the past 30 years researchers have focused on how to reduce membrane fouling. The Whitesides group has published a set of criteria for obtaining protein-resistant chemistries, viz. the surface of such membranes or materials should (i) be hydrophilic, (ii) include hydrogen bond acceptors, (iii) do not include hydrogen bond donors, and (iv) be net electrically neutral. An alternative approach to producing synthetic surfaces based on these criteria is to observe Nature’s method of producing protein-resistant surfaces. In particular, we focused on the Nuclear Pore Complex (NPC), the sole mediator of exchange between the nucleus and the cytoplasm in all eukaryotic cells. Protein transport across the NPC is fast, energy-dependent (to give directionality), often receptor-mediated and most important it occurs in the absence of unwanted fouling. For this reason we focused on “NPC-proteins” and covalently attached them on a sensor surface to test their antifouling properties. A series of different proteins, including BSA, were used to test the efficacy of this new biomimetic surface. For comparison (control), we also tested surfaces modified with other inert proteins and PEG, the gold standard with respect to protein-resistance. A quartz crystal microbalance with dissipation was used in all these experiments.
[Show abstract][Hide abstract] ABSTRACT: The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.
[Show abstract][Hide abstract] ABSTRACT: Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (∼15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with Kd values as low as ∼30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.
[Show abstract][Hide abstract] ABSTRACT: It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from non-specific interactors, preserving and isolating all unique interactions including those that are weak, transient or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact sub-micron chunks to allow for rapid access of a chemical reagent and to +stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under non-denaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry (SAC-MS). Here, we demonstrate that SAC-MS allows us to stabilize and elucidate local, distant and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.
[Show abstract][Hide abstract] ABSTRACT: Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for structural characterization of such complexes, which has at its center chemical cross-linking with mass spectrometric readout (CX-MS). In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex , a stable hetero-heptameric assembly (~600 kDa), sixteen copies of which form the outer rings of the 50 MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1-/-;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N-terminus of Nrbf2. We next used human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels as well as diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the network, leading to suppression of Vps34 activity, autophagosome biogenesis and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.
[Show abstract][Hide abstract] ABSTRACT: The nuclear pore complex (NPC) is the sole mediator of bidirectional nucleo-cytoplasmic transport and is also an important scaffold for chromatin organization and transcriptional regulation. Proteomic studies of numerous diverse eukaryotic species initially characterized the NPC as built with a number of remarkably similar structural features, suggesting its status as an ancient and conserved eukaryotic cell component. However, further detailed analyses now suggest that several key specific NPC features have a more convoluted evolutionary history than initially assumed. Recently we reported on TbNup92, a component in trypanosomes of one such conserved structural feature, a basket-like structure on the nuclear face of the NPC. We showed that TbNup92 has similar roles to nuclear basket proteins from yeasts and animals (Mlp and Tpr, respectively) in interacting with both the NPC and the mitotic spindle. However, comparative genomics suggests that TbNup92 and Mlp/Tpr may be products of distinct evolutionary histories, raising the possibility that these gene products are analogs rather than direct orthologs. Taken together with recent evidence for divergence in the nuclear lamina and kinetochores, it is apparent that the trypanosome nucleus functions by employing several novel or highly divergent protein complexes in parallel with conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of hierarchical nuclear organization.
[Show abstract][Hide abstract] ABSTRACT: The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular level of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex to be comprised of two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway.