pFind Studio: a computational solution for mass spectrometry-based proteomics
2019
Nucleic acids research2019. Han, JM et al.
Chinese Acad Sci, State Key Lab Mol Biol, Natl Ctr Prot Sci Shanghai, CAS Ctr Excellence Mol Cell Sci,Shanghai Inst Bio, 333 Haike Rd, Shanghai 201210, Peoples R China.
ABSTRACT:The Mixed Lineage Leukemia protein 1 (MLL1) plays an essential role in the maintenance of the histone H3 lysine 4 (H3K4) methylation status for gene expression during differentiation and development. The methyltransferase activity of MLL1 is regulated by three conserved core subunits, WDR5, RBBP5 and ASH2L. Here, we determined the structure of human RBBP5 and demonstrated its role in the assembly and regulation of the MLL1 complex. We identified an internal interaction between the WD40 propeller and the C-terminal distal region in RBBP5, which assisted the maintenance of the compact conformation of the MLL1 complex. We also discovered a vertebrate-specific motif in the C-terminal distal region of RBBP5 that contributed to nucleosome recognition and methylation of nucleosomes by the MLL1 complex. Our results provide new insights into functional conservation and evolutionary plasticity of the scaffold protein RBBP5 in the regulation of KMT2-family methyltransferase complexes.
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STRUCTURE2019. Prajapati, S et al.
Georg August Univ Gottingen, Gottingen Ctr Mol Biosci, Dept Mol Enzymol, Julia Lermontowa Weg 3, D-37077 Gottingen, Germany.
ABSTRACT:The pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-A resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes. Such uneven, clustered organization translates into specific duties for E1-E2 clusters (oxidative decarboxylation and acetyl transfer) and E3BP-E3 clusters (regeneration of reduced lipoamide) corresponding to half-reactions of the PDHc catalytic cycle. The addition of substrate coenzyme A modulates the conformational landscape of PDHc, in particular of the lipoyl domains, extending the postulated multiple random coupling mechanism. The conformational and associated chemical landscapes of PDHc are thus not determined entirely stochastically, but are restrained and channeled through an asymmetric architecture and further modulated by substrate binding.
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Journal of Biological Chemistry2019. Zhang, X et al.
Chinese Acad Sci, Shanghai Inst Organ Chem, Interdisciplinary Res Ctr Biol & Chem, 26 Qiuyue Rd, Shanghai 201210, Peoples R China.
ABSTRACT:Heat shock protein 104 (HSP104) is a conserved AAA+ protein disaggregase, can disassemble the toxic aggregates formed by different amyloid proteins, and is protective in various animal models associated with amyloid-related diseases. Extensive studies have attempted to elucidate how HSP104 disassembles the aggregated form of clients. Here, we found that HSP104 exhibits a potent holdase activity that does not require energy, prevents the soluble form of amyloid clients from aggregating, and differs from HSP104's disaggregase activity. Using cryo-EM, NMR, and additional biophysical approaches, we found that HSP104 utilizes its small subdomain of nucleotide-binding domain 2 (ssNBD2) to capture the soluble amyloid client (K19 of Tau) independent of its ATP hydrolysis activity. Our results indicate that HSP104 utilizes two fundamental distinct mechanisms to chaperone different forms of amyloid client and highlight the important yet previously unappreciated function of ssNBD2 in chaperoning amyloid client and thereby preventing pathological aggregation.
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PLOS Pathogens2019. Mirzakhanyan, Y et al.
UC Irvine, Dept Mol Biol & Biochem, Irvine, CA 92697 USA.
ABSTRACT:We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an attachment protein' sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus. Author summary Vaccinia is one of the most complex virions among the animal viruses, containing 70+ distinct gene products. Although virion ultrastructure has been apparent, at least in outline by electron microscopy since the year 1961 or earlier, its molecular architecture is largely unknown: Vaccinia is resistant to classical structural approaches requiring virus crystallization and moderately resistant to cryoEM. Molecular approaches requiring the maintenance of protein assemblies during virion deconstruction, reconstruction of protein complexes in heterologous or in vitro systems, or internalization of bulky reagents such as antibodies or gold particles may have been already pursued close to exhaustion. Here, protein interfaces within and around the intact virion were identified by virus incubation with bifunctional chemical crosslinkers in situ followed by proteolysis and peptide-level mass spectrometry. This minimally invasive approach revealed the molecular arrangements of structural and membrane protein complexes within the virus, confirming and extending several aspects of virus biology.
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Nutrients2019. Lexhaller, B et al.
Tech Univ Munich, Leibniz Inst Food Syst Biol, Lise Meitner Str 34, D-85354 Freising Weihenstephan, Germany.
ABSTRACT:Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in CD pathogenesis, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent N epsilon-(gamma-glutamyl)-lysine isopeptide crosslinks resulting in TG2-gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we used different proteomics software tools that enable the comprehensive identification of isopeptides. Thus, 34 different isopeptides involving 20 TG2 lysine residues were identified in a model system, only six of which were previously known. Additionally, 36 isopeptides of TG2-TG2 multimers were detected. Experiments with different TG2-gluten peptide molar ratios revealed the most preferred lysine residues involved in isopeptide crosslinking. Expanding the model system to three gluten peptides with more glutamine residues allowed the localization of the preferred glutamine crosslinking sites. These new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.
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Journal of proteome research2019. Fang, ZX et al.
Univ Texas Arlington, Dept Chem & Biochem, Arlington, TX 76019 USA.
ABSTRACT:Chemical cross-linking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for cross-linking/MS is the determination of informative low abundant inter-cross-linked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reversed-phase (RP) separation of inter-cross-linked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases. In this study, two phenyl-based columns, biphenyl and fluorophenyl, were investigated and compared with a C18 phase for separating BS3 (bis(sulfosuccinimidyl)suberate) cross-linked bovine serum albumin (BSA) and myoglobin by bottom-up proteomics. Fractions from the three columns were collected and analyzed in a linear ion trap (LIT) mass spectrometer for improving detection of low abundant inter-cross-linked peptides. Among these three columns, the fluorophenyl column provides additional ion-exchange interaction and exhibits unique retention in separating the cross-linked peptides. The fractioned data was analyzed in pLink, showing the fluorophenyl column consistently obtained more inter-cross-linked peptide identifications than both C18 and biphenyl columns. For the BSA cross-linked sample, the identified inter-cross-linked peptide numbers of the fluorophenyl to C18 column are 136 to 102 in "low confident" results and 11 to 6 in "high confident" results. The fluorophenyl column could potentially be a better alternative for targeting the low stoichiometric inter-cross-linked peptides.
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Biochemical and biophysical research communications2019. Wu, H et al.
Chinese Acad Sci, Shanghai Inst Appl Phys, Shanghai 201800, Peoples R China.
ABSTRACT:Quorum sensing regulates the biofilm formation and expression of virulence factors in Vibrio cholerae, an obligate human pathogen that continues to imperil human health. Cytoplasmic transcription factor VqmA is a LuxR-type receptor ubiquitous in the Vibrio genus and one vibriophage VP882 and plays an important role in V. cholerae pathogenicity. Here we presented the X-ray crystal structure of V. cholerae VqmA-DPO complex and compared it with the previously determined VqmA-DPO-DNA complex. To our knowledge, this is the first report on the crystal structures of the same LuxR-type receptor with two conformations of binding to DNA and not binding to DNA. Based on the results of structural analysis and biochemical assays, we revealed the secondary structure of the linker region between two function domains changed significantly, and DNA binding domains were covalently linked by a disulfide bond formed by the highly conserved Cys134. Besides, the distance between two DBD monomers became longer than that in DNA-binding conformation, and two alpha 8 helixes underwent a large conformation shift. The results of the structure-function analyses presented here improve our understanding of the complex mechanisms in the conformational changes of LuxR-type receptors caused by DNA binding. (C) 2019 Elsevier Inc. All rights reserved.
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Bioinformatics2019. Dai, JA et al.
Hong Kong Univ Sci & Technol, Dept Elect & Comp Engn, Hong Kong, Peoples R China.
ABSTRACT:Motivation: Cross-linking technique coupled with mass spectrometry (MS) is widely used in the analysis of protein structures and protein-protein interactions. In order to identify cross-linked peptides from MS data, we need to consider all pairwise combinations of peptides, which is computationally prohibitive when the sequence database is large. To alleviate this problem, some heuristic screening strategies are used to reduce the number of peptide pairs during the identification. However, heuristic screening strategies may miss some true cross-linked peptides. Results: We directly tackle the combination challenge without using any screening strategies. With the data structure of double-ended queue, the proposed algorithm reduces the quadratic time complexity of exhaustive searching down to the linear time complexity. We implement the algorithm in a tool named Xolik. The running time of Xolik is validated using databases with different numbers of proteins. Experiments using synthetic and empirical datasets show that Xolik outperforms existing tools in terms of running time and statistical power.
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Cellular and Molecular Life Sciences2019. Wu, JW et al.
Shanghai Jiao Tong Univ, Sch Med, Dept Pathophysiol, Shanghai Tongren Hosp,Fac Basic Med,Hongqiao Int, Shanghai 200025, Peoples R China.
ABSTRACT:The P3H1/CRTAP/PPIB complex is essential for prolyl 3-hydroxylation and folding of procollagens in the endoplasmic reticulum (ER). Deficiency in any component of this ternary complex is associated with the misfolding of collagen and the onset of osteogenesis imperfecta. However, little structure information is available about how this ternary complex is assembled and retained in the ER. Here, we assessed the role of the KDEL sequence of P3H1 and probed the spatial interactions of PPIB in the complex. We show that the KDEL sequence is essential for retaining the P3H1 complex in the ER. Its removal resulted in co-secretion of P3H1 and CRTAP out of the cell, which was mediated by the binding of P3H1 N-terminal domain with CRTAP. The secreted P3H1/CRTAP can readily bind PPIB with their C-termini close to PPIB in the ternary complex. Cysteine modification, crosslinking, and mass spectrometry experiments identified PPIB surface residues involved in the complex formation, and showed that the surface of PPIB is extensively covered by the binding of P3H1 and CRTAP. Most importantly, we demonstrated that one disease-associated pathological PPIB mutation on the binding interface did not affect the PPIB prolyl-isomerase activity, but disrupted the formation of P3H1/CRTAP/PPIB ternary complex. This suggests that defects in the integrity of the P3H1 ternary complex are associated with pathological collagen misfolding. Taken together, these results provide novel structural information on how PPIB interacts with other components of the P3H1 complex and indicate that the integrity of P3H1 complex is required for proper collagen formation.
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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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