pFind Studio: a computational solution for mass spectrometry-based proteomics
2015
Journal of Biological chemistry2015. LoPiccolo, J et al.
Albert Einstein Coll Med, Dept Biochem, 1300 Morris Pk Ave, Bronx, NY 10461 USA.
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. p85 alpha, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85 alpha 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 p85 alpha that is functionally equivalent to native p85 alpha. Analytical ultracentrifugation studies showed that p85 alpha 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. We have demonstrated in vivo concentration-dependent dimerization of p85 alpha using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85 alpha dimer. Our study provides new insight into the structure and assembly of the p85 alpha homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.
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Biophysics reports2015. Gong, Zhou et al.
CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, China
ABSTRACT:GRAPHICAL Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-mol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure. We further show that the seemingly incompatible cross-links identified with high confidence arise from alternative modes of protein-protein interactions. By converting the intermolecular cross-links to ambiguous distance restraints, we established a rigid-body simulated annealing refinement protocol to seek the minimum set of conformers collectively satisfying the CXMS data. Hence we demonstrate that CXMS allows the depiction of the ensemble structures of protein complexes and elucidates the interaction dynamics for transient and fleeting complexes.
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GENES & DEVELOPMENT2015. Kapoor, P et al.
Univ Texas MD Anderson Canc Ctr, Dept Epigenet & Mol Carcinogenesis, Smithville, TX 78957 USA.
ABSTRACT:ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity. In vivo, Mec1 activity is reduced by the deletion of Snf2, the core ATPase subunit of the SWI/SNF complex. SWI/SNF interacts with Mec1, and cross-linking studies revealed that the Snf2 ATPase is the main interaction partner for Mec1. In vitro, SWI/SNF can activate Mec1 kinase activity in the absence of chromatin or known activators such as Dpb11. The subunit requirement of SWI/SNF-mediated Mec1 regulation differs from that of SWI/SNF-mediated chromatin remodeling. Functionally, SWI/SNF-mediated Mec1 regulation specifically occurs in S phase of the cell cycle. Together, these findings identify a novel regulator of Mec1 kinase activity and suggest that ATP-dependent chromatin remodeling complexes can regulate nonchromatin substrates such as a checkpoint kinase.
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Protein Science2015. Nguyen-Huynh, NT et al.
Univ Strasbourg Chim Matiere Complexe, LSMIS, UMR CNRS 7140, 1 Rue Blaise Pascal, F-67008 Strasbourg, France.
ABSTRACT:Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.
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Journal of Biological chemistry2015. Reimund, M et al.
Akad Tee 15, EE-12618 Tallinn, Estonia.
ABSTRACT:GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on-and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.
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