pFind Studio: a computational solution for mass spectrometry-based proteomics
2019
Journal of Biological Chemistry2019. Ludlam, WG et al.
Brigham Young Univ, Dept Chem & Biochem, Provo, UT 84602 USA.
ABSTRACT:Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the ?-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its ?-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation.
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The Federation of European Biochemical Societies Journal2019. Yin, XY et al.
Univ Sci & Technol China, Med Ctr, Hefei, Anhui, Peoples R China.
ABSTRACT:Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 angstrom resolutions, respectively. The mouse DR3 DD adopts a classical six-helix bundle structure while human DR3 DD displays an extended fold. Though there is one-amino-acid difference in the linker between maltose-binding protein (MBP) tag and DR3 DD, according to our self-interaction analysis, the hydrophobic interface discovered in MBP-hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction. Furthermore, our biochemical analysis indicates that the sequence variation between human and mouse DR3 DD does not affect its structure and function. Small-angle X-ray scattering analysis shows the averaged solution structures of both human and mouse MBP-DR3 DD are the combination of different conformations with different proportion. Through switching to the open conformation, DR3 DD could improve the interaction with downstream element TNFR-associated death domain (TRADD). Here we propose an activation-dependent structural rearrangement model: the DD region is folded as the six-helix bundles in the resting state, while upon extracellular ligand engagement, it switches to the open conformation, which facilitates its self-association and the recruitment of TRADD. Our results provide detailed insights into the architecture of DR3 DD and the molecular mechanism of activation. Databases All refined structure coordinates as well as the corresponding structure factors have been deposited in the PDB under the accession codes , , , , .
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ACS Chemical Biology2019. Zheng, QZ et al.
Tsinghua Univ, Dept Chem, Ctr Basic Mol Sci, Beijing 100084, Peoples R China.
ABSTRACT:Abscisic acid (ABA) is a key phytohormone with diverse functions in plants, and its signal transduction is mainly mediated by ABA receptors termed PYR/PYL/RCARs (hereafter referred to as PYLs) through the PYLs-PP2Cs-SnRK2s regulatory systems. However, the model failed to account for the roles of some important known regulators of ABA physiology. Given the central role of PYLs in ABA signal transduction, we therefore speculated that ABA receptors PYLs might be involved in regulatory pathways other than PP2Cs. Thus, a comprehensive analysis of PYL5-interacting partners could greatly facilitate the identification of unknown regulatory pathways, advancing our knowledge of the ABA signaling mechanism. Herein, we present a strategy involving covalent chemical capture coupled with HPLC-MS/MS analysis, to profile PYL5-interacting partners in plant cell lysates. With this strategy, three new PYL5-interacting partners, ubiquitin receptor RAD23C, COP9 signalosome complex subunit 1 (CSN1), and cyclase-associated protein 1 (CAP1), along with their key binding sites with PYL5 were identified. Among these proteins, CAP1 was verified to interact with PYL5 both in vitro and in vivo. The discovery of a new PYL5 binding partner showed the versatility of covalent chemical cross-linking and laid the foundation for future efforts to further elucidate the ABA signaling mechanism.
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Journal of The American Society for Mass Spectrometry2019. Luo, J et al.
Inst Syst Biol, 401 Terry Ave North, Seattle, WA 98109 USA.
ABSTRACT:Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein-protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information. Here we introduce a novel class of MS cleavable cross-linkers called isotopomeric cross-linkers (ICLs), which allow for confident and efficient identification of cross-linked peptides by whole proteome database searching. ICLs are simple, symmetrical molecules that asymmetrically incorporate heavy and light stable isotopes into the two arms of the cross-linker. As a result of this property, ICLs automatically generate pairs of isotopomeric cross-linked peptides, which differ only by the positions of the heavy and light isotopes. Upon fragmentation during MS analysis, these isotopomeric cross-linked peptides generate unique isotopic doublet ions that correspond to the individual peptides in the cross-link. The doublet ion information is used to determine the masses of the two cross-linked peptides from the same MS2 spectrum that is also used for peptide spectrum matching (PSM) by sequence database searching. Here we present the rationale for and mechanism of cross-linked peptide identification by ICL-MS. We describe the synthesis of the ICL-1 reagent, the ICL-MS workflow, and the performance characteristics of ICL-MS for identifying cross-linked peptides derived from increasingly complex mixtures by whole proteome database searching.
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Journal of Biological Chemistry2019. Roy, NS et al.
NCI, Lab Cellular & Mol Biol, Ctr Canc Res, NIH, Frederick, MD 21702 USA.
ABSTRACT:Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Delta 17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP(2)), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Delta 17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first beta-strand of the PH domain. Point mutations in either the linker or the C-terminal alpha-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
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Analytical Chemistry2019. Zhang, MM et al.
Washington Univ, Dept Chem, St Louis, MO 63130 USA.
ABSTRACT:We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its alpha-receptor, IL-7R alpha, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7R alpha but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7R alpha complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 angstrom relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.
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Analytical Chemistry2019. Zhao, LL et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China.
ABSTRACT:Chemical cross-linking combined with mass spectrometry (CXMS) has emerged as a powerful tool to study protein structure, conformation, and protein-protein interactions (PPIs). Until now, most cross-linked peptides were generated by using commercial cross-linkers, such as DSS, BS3, and DSSO, which react with the primary amino groups of the lysine residues of proteins. However, trypsin, the most commonly used proteolytic enzyme, cannot cleave the C-terminus of a linked lysine, making the obtained cross-linked peptides longer than common peptides and unfavorable for MS identification and data searching. Herein, we propose an in situ sequential digestion strategy using enzymes with distinct cleavage specificity, named as smart cutter, to generate cross-linked peptides with suitable length so that the identification coverage could improve. Through the application of such a strategy to DSS cross-linked E. coli lysates, additional cross-linked sites (1.3-fold increase) obtained in comparison with those obtained by trypsin-trypsin digestion (2879 vs 1255). Among the different digestion combinations, AspN-trypsin performed the best, with 64% (673/1059) of the cross-linked sites complementary to trypsin-trypsin digestion, which is beneficial to ensure the depth for studying protein structure and PPIs. Taking the 60 kDa chaperonin protein as an example, more than twice the cross-linked sites (30 vs 14) were identified to enrich the protein structure information. In addition, compared to the published protein interaction network for E. coli (http://www.bacteriome.org), 91 potential PPIs were discovered with our strategy, of which 65 have not covered by trypsin-trypsin digestion. Therefore, these results illustrate the great significance of smart-cutter-based CXMS for the revelation of protein structure as well as finding new PPIs.
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Journal of Biological Chemistry2019. Schmitt, LR et al.
Univ Colorado, Sch Med, Dept Biochem & Mol Genet, Aurora, CO 80045 USA.
ABSTRACT:The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the C region of fibrinogen . Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.
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Molecular Cell2019. Valencia-Sanchez, MI et al.
NYU, Sch Med, Dept Biochem & Mol Pharmacol, Skirball Inst Biomol Med, New York, NY 10016 USA.
ABSTRACT:The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Doti L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 angstrom and 4.9 angstrom, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.
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Journal of Agricultural and Food Chemistry2019. Sheng, JY et al.
Huazhong Univ Sci & Technol, Coll Life Sci & Technol, Natl Engn Res Ctr Nanomed, Wuhan 430074, Hubei, Peoples R China.
ABSTRACT:The peptide components of defatted walnut (Juglans regia L.) meal hydrolysate (DWMH) remain unclear, hindering the investigation of biological mechanisms and exploitation of bioactive peptides. The present study aims to identify the peptide composition of DWMH, followed by to evaluate in vitro antioxidant effects of selected peptides and investigate mechanisms of antioxidative effect. First, more than 1 000 peptides were identified by de novo sequencing in DWMH. Subsequently, a scoring method was established to select promising bioactive peptides by structure based screening. Eight brand new peptides were selected due to their highest scores in two different batches of DWMH. All of them showed potent in vitro antioxidant effects on H2O2-injured nerve cells. Four of them even possessed significantly stronger effects than DWMH, making the selected bioactive peptides useful for further research as new bioactive entities. Two mechanisms of hydroxyl radical scavenging and ROS reduction were involved in their antioxidative effects at different degrees. The results showed peptides possessing similar capacity of hydroxyl radical scavenging or ROS reduction may have significantly different in vitro antioxidative effects. Therefore, comprehensive consideration of different antioxidative mechanisms were suggested in selecting antioxidative peptides from DWMH.
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