Mapping low-affinity/high-specificity peptide–protein interactions using ligand-footprinting mass spectrometry

Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide–mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures. LiF-MS peptide ligands carry a short-lived, indiscriminately reactive cleavable crosslinker that marks contacts close to ligand binding sites with high specificity. Each marked amino acid provides an independent constraint for a set of directed peptide–protein docking simulations, which are analyzed by agglomerative hierarchical clustering. We found that LiF-MS provides accurate ab initio identification of ligand binding surfaces and a view of potential binding ensembles of a set of D-motif peptide–MAPK associations. Our analysis provides an MKK4–JNK1 structural model, which has thus far been crystallographically unattainable, a potential alternate binding mode for part of the NFAT4–JNK interaction, and evidence of bidirectional association of MKK4 peptide with ERK2. Overall, we find that LiF-MS is an effective noncrystallographic way to understand how short linear motifs associate with specific sites on folded protein domains at the level of individual amino acids.

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Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

Biochemical Journal ◽

10.1042/bj2850419 ◽

1992 ◽

Vol 285 (2) ◽

pp. 419-425 ◽

Cited By ~ 34

Author(s):

U Christensen ◽

L Mølgaard

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Conformational Change ◽

Conformational Changes ◽

Binding Sites ◽

High Specificity ◽

Stopped Flow ◽

Protein Fluorescence ◽

Lysine Residues ◽

Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

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Probing ligand and cation binding sites in G-quadruplex nucleic acids by mass spectrometry and electron photodetachment dissociation sequencing

The Analyst ◽

10.1039/c9an00398c ◽

2019 ◽

Vol 144 (11) ◽

pp. 3518-3524 ◽

Cited By ~ 6

Author(s):

Dababrata Paul ◽

Adrien Marchand ◽

Daniela Verga ◽

Marie-Paule Teulade-Fichou ◽

Sophie Bombard ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Ligand Binding ◽

Binding Sites ◽

Cation Binding ◽

Tandem Mass ◽

Top Down ◽

G Quadruplex ◽

Ligand Binding Sites ◽

Electron Photodetachment

Tandem mass spectrometry: native top-down sequencing by electron photodetachment dissociation (EPD) reveals ligand binding sites on DNA G-quadruplexes.

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A structural model for the ligand-binding sites at the nicotinic acetylcholine receptor

FEBS Letters ◽

10.1016/0014-5793(84)81241-7 ◽

1984 ◽

Vol 178 (1) ◽

pp. 64-68 ◽

Cited By ~ 29

Author(s):

Louis Smart ◽

Hans-Wilhelm Meyers ◽

Rolf Hilgenfeld ◽

Wolfram Saenger ◽

Alfred Maelicke

Keyword(s):

Ligand Binding ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Binding Sites ◽

Structural Model ◽

Nicotinic Acetylcholine ◽

Ligand Binding Sites

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Protein docking and steered molecular dynamics suggest alternative phospholamban-binding sites on the SERCA calcium transporter

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012948 ◽

2020 ◽

Vol 295 (32) ◽

pp. 11262-11274

Author(s):

Rebecca F. Alford ◽

Nikolai Smolin ◽

Howard S. Young ◽

Jeffrey J. Gray ◽

Seth L. Robia

Keyword(s):

Molecular Dynamics ◽

Binding Sites ◽

Structural Model ◽

Steered Molecular Dynamics ◽

Protein Docking ◽

Structural Determinants ◽

Inhibitory Interaction ◽

Physical Measurements ◽

Inhibitory Site ◽

Regulatory Complex

The transport activity of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) in cardiac myocytes is modulated by an inhibitory interaction with a transmembrane peptide, phospholamban (PLB). Previous biochemical studies have revealed that PLB interacts with a specific inhibitory site on SERCA, and low-resolution structural evidence suggests that PLB interacts with distinct alternative sites on SERCA. High-resolution details of the structural determinants of SERCA regulation have been elusive because of the dynamic nature of the regulatory complex. In this study, we used computational approaches to develop a structural model of SERCA–PLB interactions to gain a mechanistic understanding of PLB-mediated SERCA transport regulation. We combined steered molecular dynamics and membrane protein–protein docking experiments to achieve both a global search and all-atom force calculations to determine the relative affinities of PLB for candidate sites on SERCA. We modeled the binding of PLB to several SERCA conformations, representing different enzymatic states sampled during the calcium transport catalytic cycle. The results of the steered molecular dynamics and docking experiments indicated that the canonical PLB-binding site (comprising transmembrane helices M2, M4, and M9) is the preferred site. This preference was even more stringent for a superinhibitory PLB variant. Interestingly, PLB-binding specificity became more ambivalent for other SERCA conformers. These results provide evidence for polymorphic PLB interactions with novel sites on M3 and with the outside of the SERCA helix M9. Our findings are compatible with previous physical measurements that suggest that PLB interacts with multiple binding sites, conferring dynamic responsiveness to changing physiological conditions.

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Bioinformatic and biochemical analysis of the key binding sites of the pheromone binding protein of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidea)

PeerJ ◽

10.7717/peerj.7818 ◽

2019 ◽

Vol 7 ◽

pp. e7818 ◽

Cited By ~ 3

Author(s):

Hua Yang ◽

Yan-Lin Liu ◽

Yuan-Yuan Tao ◽

Wei Yang ◽

Chun-Ping Yang ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Binding Sites ◽

Dibutyl Phthalate ◽

Structural Model ◽

Site Directed Mutagenesis ◽

Economic Losses ◽

Directed Mutagenesis ◽

Wide Range ◽

Cyrtotrachelus Buqueti

The bamboo snout beetle Cyrtotrachelus buqueti is a widely distributed wood-boring pest found in China, and its larvae cause significant economic losses because this beetle targets a wide range of host plants. A potential pest management measure of this beetle involves regulating olfactory chemoreceptors. In the process of olfactory recognition, pheromone-binding proteins (PBPs) play an important role. Homology modeling and molecular docking were conducted in this study for the interaction between CbuqPBP1 and dibutyl phthalate to better understand the relationship between PBP structures and their ligands. Site-directed mutagenesis and binding experiments were combined to identify the binding sites of CbuqPBP1 and to explore its ligand-binding mechanism. The 3D structural model of CbuqPBP1 has six a-helices. Five of these a-helices adopt an antiparallel arrangement to form an internal ligand-binding pocket. When docking dibutyl phthalate within the active site of CbuqPBP1, a CH-π interaction between the benzene ring of dibutyl phthalate and Phe69 was observed, and a weak hydrogen bond formed between the ester carbonyl oxygen and His53. Thus, Phe69 and His53 are predicted to be important residues of CbuqPBP1 involved in ligand recognition. Site-directed mutagenesis and fluorescence assays with a His53Ala CbuqPBP1 mutant showed no affinity toward ligands. Mutation of Phe69 only affected binding of CbuqPBP1 to cedar camphor. Thus, His53 (Between α2 and α3) of CbuqPBP1 appears to be a key binding site residue, and Phe69 (Located at α3) is a very important binding site for particular ligand interactions.

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Probing Ligand and Cation Binding Sites in G-Quadruplex Nucleic Acids by Mass Spectrometry and Electron Photodetachment Dissociation Sequencing

10.1101/563627 ◽

2019 ◽

Author(s):

Dababrata Paul ◽

Adrien Marchand ◽

Daniela Verga ◽

Marie-Paule Teulade-Fichou ◽

Sophie Bombard ◽

...

Keyword(s):

Mass Spectrometry ◽

Ligand Binding ◽

Binding Sites ◽

Binding Mode ◽

Cation Binding ◽

Potassium Cation ◽

Telomeric Sequences ◽

G Quadruplex ◽

Electron Photodetachment ◽

Noncovalent Bonds

ABSTRACTMass spectrometry provides exquisite detail on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by the mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By obtaining more negative charge states for the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

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Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry

Journal of the American Society for Mass Spectrometry ◽

10.1007/s13361-018-1998-7 ◽

2018 ◽

Vol 29 (9) ◽

pp. 1881-1891 ◽

Cited By ~ 1

Author(s):

Yannick Baschung ◽

Loredana Lupu ◽

Adrian Moise ◽

Michael Glocker ◽

Stephan Rawer ◽

...

Keyword(s):

Mass Spectrometry ◽

Ligand Binding ◽

Blood Group ◽

Binding Sites ◽

Ligand Binding Sites

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Targeted Site-Specific Gas-Phase Cleavage of Oligoribonucleotides. Application in Mass Spectrometry-Based Identification of Ligand Binding Sites

Journal of the American Chemical Society ◽

10.1021/ja983131x ◽

1999 ◽

Vol 121 (2) ◽

pp. 474-475 ◽

Cited By ~ 31

Author(s):

Richard H. Griffey ◽

Michael J. Greig ◽

Haoyun An ◽

Henri Sasmor ◽

Sherilynn Manalili

Keyword(s):

Mass Spectrometry ◽

Ligand Binding ◽

Gas Phase ◽

Binding Sites ◽

Site Specific ◽

Ligand Binding Sites

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Top-Down ESI-ECD-FT-ICR Mass Spectrometry Localizes Noncovalent Protein-Ligand Binding Sites

Journal of the American Chemical Society ◽

10.1021/ja063197p ◽

2006 ◽

Vol 128 (45) ◽

pp. 14432-14433 ◽

Cited By ~ 142

Author(s):

Yongming Xie ◽

Jennifer Zhang ◽

Sheng Yin ◽

Joseph A. Loo

Keyword(s):

Mass Spectrometry ◽

Ligand Binding ◽

Binding Sites ◽

Top Down ◽

Ligand Binding Sites ◽

Noncovalent Protein ◽

Ft Icr

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Identification of ligand binding sites in intrinsically disordered proteins with a differential binding score

Scientific Reports ◽

10.1038/s41598-021-00869-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Qiao-Hong Chen ◽

V. V. Krishnan

Keyword(s):

Drug Discovery ◽

Ligand Binding ◽

Binding Sites ◽

Intrinsically Disordered Proteins ◽

Random Coil ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Binding Score ◽

Ligand Binding Sites ◽

Differential Binding

AbstractScreening ligands directly binding to an ensemble of intrinsically disordered proteins (IDP) to discover potential hits or leads for new drugs is an emerging but challenging area as IDPs lack well-defined and ordered 3D-protein structures. To explore a new IDP-based rational drug discovery strategy, a differential binding score (DIBS) is defined. The basis of DIBS is to quantitatively determine the binding preference of a ligand to an ensemble of conformations specified by IDP versus such preferences to an ensemble of random coil conformations of the same protein. Ensemble docking procedures performed on repeated sampling of conformations, and the results tested for statistical significance determine the preferential ligand binding sites of the IDP. The results of this approach closely reproduce the experimental data from recent literature on the binding of the ligand epigallocatechin gallate (EGCG) to the intrinsically disordered N-terminal domain of the tumor suppressor p53. Combining established approaches in developing a new method to screen ligands against IDPs could be valuable as a screening tool for IDP-based drug discovery.

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