scholarly journals Integrating cross-linking experiments with ab initio protein-protein docking

2018 ◽  
Author(s):  
Thom Vreven ◽  
Devin K. Schweppe ◽  
Juan D. Chavez ◽  
Chad R. Weisbrod ◽  
Sayaka Shibata ◽  
...  
Keyword(s):  
Experimental Data ◽  
Ab Initio ◽  
Structure Prediction ◽  
Complex Structure ◽  
Protein Docking ◽  
Cross Linking ◽  
Test Cases ◽  
List Type ◽  
Cross Links ◽  
Chemical Cross Linking

ABSTRACTAb initio protein-protein docking algorithms often rely on experimental data to identify the most likely complex structure. We integrated protein-protein docking with the experimental data of chemical cross-linking followed by mass spectrometry. We tested our approach using 12 cases that resulted from an exhaustive search of the Protein Data Bank for protein complexes with cross-links identified in our experiments. We implemented cross-links as constraints based on Euclidean distance or void-volume distance. For most test cases the rank of the top-scoring near-native prediction was improved by at least two fold compared with docking without the cross-link information, and the success rates for the top 5 and top 10 predictions doubled. Our results demonstrate the delicate balance between retaining correct predictions and eliminating false positives. Several test cases had multiple components with distinct interfaces, and we present an approach for assigning cross-links to the interfaces. Employing the symmetry information for these cases further improved the performance of complex structure prediction.HighlightsIncorporating low-resolution cross-linking experimental data in protein-protein docking algorithms improves performance more than two fold.Integration of protein-protein docking with chemical cross-linking reveals information on the configuration of higher order complexes.Symmetry analysis of protein-protein docking results improves the predictions of multimeric complex structures

scholarly journals ResDock: Protein-Protein Docking Using Shape Complementarity of Surface Residues

2021 ◽  
Author(s):  
Sharon Sunny ◽  
Jayaraj PB
Keyword(s):  
Ab Initio ◽  
Protein Complex ◽  
Structure Prediction ◽  
Complex Structure ◽  
Scoring Function ◽  
Protein Docking ◽  
New Method ◽  
Protein Surfaces ◽  
Shape Complementarity ◽  
Conformation Space

ResDock is a new method to improve the performance of protein-protein complex structure prediction. It utilizes shape complementarity of the protein surfaces to generate the conformation space. The use of an appropriate scoring function helps to select the feasible structures. An interplay between pose generation phase and scoring phase enhance the performance of the proposed ab initio technique. <br>

scholarly journals ResDock: Protein-Protein Docking Using Shape Complementarity of Surface Residues

2021 ◽  
Author(s):  
Sharon Sunny ◽  
Jayaraj PB
Keyword(s):  
Ab Initio ◽  
Protein Complex ◽  
Structure Prediction ◽  
Complex Structure ◽  
Scoring Function ◽  
Protein Docking ◽  
New Method ◽  
Protein Surfaces ◽  
Shape Complementarity ◽  
Conformation Space

ResDock is a new method to improve the performance of protein-protein complex structure prediction. It utilizes shape complementarity of the protein surfaces to generate the conformation space. The use of an appropriate scoring function helps to select the feasible structures. An interplay between pose generation phase and scoring phase enhance the performance of the proposed ab initio technique. <br>

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

A Geometric Complementarity-Based Tool for Protein–Protein Docking

2021 ◽  
Author(s):  
Sharon Sunny ◽  
P. B. Jayaraj
Keyword(s):  
Structure Prediction ◽  
Complex Structure ◽  
Protein Docking ◽  
Geometric Algorithms ◽  
Coarse Grained ◽  
Biological Impact ◽  
Searching Strategy ◽  
Determining Factor ◽  
Docking Benchmark ◽  
Conformation Space

The computationally hard protein–protein complex structure prediction problem is continuously fascinating to the scientific community due to its biological impact. The field has witnessed the application of geometric algorithms, randomized algorithms, and evolutionary algorithms to name a few. These techniques improve either the searching or scoring phase. An effective searching strategy does not generate a large conformation space that perhaps demands computational power. Another determining factor is the parameter chosen for score calculation. The proposed method is an attempt to curtail the conformations by limiting the search procedure to probable regions. In this method, partial derivatives are calculated on the coarse-grained representation of the surface residues to identify the optimal points on the protein surface. Contrary to the existing geometric-based algorithms that align the convex and concave regions of both proteins, this method aligns the concave regions of the receptor with convex regions of the ligand only and thus reduces the size of conformation space. The method’s performance is evaluated using the 55 newly added targets in Protein–Protein Docking Benchmark v 5 and is found to be successful for around 47% of the targets.

Abstract 263: Isotopic Labeling and Chemical Cross-Linking Reveals Intermolecular Contacts and Dynamics of Apolipoprotein A-I Structure in Reconstituted HDL Particles

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamie Morris ◽  
Martin K Jones ◽  
Gang Ren ◽  
Jere Segrest ◽  
W Sean Davidson
Keyword(s):  
Conformational Dynamics ◽  
Density Lipoprotein ◽  
Isotopic Labeling ◽  
Peptide Fragmentation ◽  
Cross Linking ◽  
Naturally Occurring ◽  
Intermolecular Contacts ◽  
Cross Links ◽  
Mass Shifts ◽  
Chemical Cross Linking

Apolipoprotein (apo)A-I has been proposed to adopt a number of different, but related, structures when in contact with lipid. The technique of chemical cross-linking has recently been used to determine the spatial relationships between two molecules of apoA-I in reconstituted high density lipoprotein (rHDL) particles. However, this technique is limited in that it cannot unequivocally distinguish between intramolecular and intermolecular contacts. To address this issue, we have produced two forms of recombinant apoA-I that contain the naturally occurring isotope of nitrogen (N14) or a stable isotope (N15). These forms were mixed 1:1 and then used to produce reconstituted HDL particles with synthetic lipids. The resulting mass shifts (readily detectable in the mass spectrometer) were exploited to unambiguously distinguish between intramolecular (N14 to N14 or N15 to N15) and intermolecular (N14 to N15) cross-linked peptides. An additional benefit of this approach was the ability to identify cross-links with high certainty without the need for peptide fragmentation, allowing for dramatic increases in method sensitivity. We studied highly homogeneous rHDL particles made with the fully saturated phospholipid palmitoyl steroyl phosphatidylcholine (PSPC) to minimize apoA-I conformational dynamics. These particles were 98Å in diameter, contained two molecules of apoA-I, approximately 155 molecules of PC, and were discoidal in shape by cryo EM. We identified 30 cross-links (17 intramolecular, 13 intermolecular) that were overall consistent with the double belt model in which both apoA-I molecules wrap around a bilayer of lipids in an antiparallel orientation. Unambiguous intramolecular linkages between the N- and C-terminal regions of apoA-I were inconsistent with the ‘double super helix’ variant of the belt model. We identified several low abundance cross-links that suggest that the N-terminus may be conformationally dynamic and may spend some time folded back across the molecule as proposed for the ‘belt and buckle’ belt model.

scholarly journals High-density chemical cross-linking for modeling protein interactions

2019 ◽  
Vol 117 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Julian Mintseris ◽  
Steven P. Gygi
Keyword(s):  
Mass Spectrometry ◽  
Method Development ◽  
Conformational Dynamics ◽  
Complex Function ◽  
Computational Cost ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Proteasome Subunits ◽  
Cross Links ◽  
Chemical Cross Linking

Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are self-consistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique cross-linked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.

scholarly journals Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

2017 ◽  
Vol 114 (7) ◽  
pp. 1732-1737 ◽  
Author(s):  
Devin K. Schweppe ◽  
Juan D. Chavez ◽  
Chi Fung Lee ◽  
Arianne Caudal ◽  
Shane E. Kruse ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Mitochondrial Function ◽  
Mitochondrial Protein ◽  
Protein Structures ◽  
Protein Docking ◽  
Molecular Probes ◽  
Cross Linking ◽  
Chemical Cross Linking

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I–III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

scholarly journals Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lisa Rebers ◽  
Raffael Reichsöllner ◽  
Sophia Regett ◽  
Günter E. M. Tovar ◽  
Kirsten Borchers ◽  
...  
Keyword(s):  
Near Infrared ◽  
Cross Linking ◽  
Cross Link ◽  
Scanning Calorimetry ◽  
Link Density ◽  
Double Bond Conversion ◽  
Cross Links ◽  
Physical And Chemical ◽  
The Impact ◽  
Chemical Cross Linking

AbstractGelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.

scholarly journals Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Carla Schmidt ◽  
Min Zhou ◽  
Hazel Marriott ◽  
Nina Morgner ◽  
Argyris Politis ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Mass Spectra ◽  
Nucleotide Binding ◽  
Cross Linking ◽  
Spinach Chloroplasts ◽  
Cross Links ◽  
Chemical Cross Linking

Abstract F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid ‘plug’ in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.

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