TY - JOUR
T1 - Mapping low-affinity/high-specificity peptide–protein interactions using ligand-footprinting mass spectrometry
AU - Parker, Benjamin W.
AU - Goncz, Edward J.
AU - Krist, David T.
AU - Statsyuk, Alexander V.
AU - Nesvizhskii, Alexey I.
AU - Weiss, Eric L.
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Felipe da Veiga Leprevost for help with MS/MS data analysis, Gergo} Gógl and Attila Reményi for reagents, and Jennifer Brace for critical review and editing of the manuscript. This research was supported by the National Institute of General Medical Sciences of the NIH grant R01GM084223 to E.L.W. and in part by NIH grants R01GM94231 and U24CA210967 to A.I.N. This research used facilities supported by National Cancer Institute CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center (Northwestern University Proteomics Core Facility and Structural Biology Facility) and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569 (Northwestern University Proteomics Core Facility).
Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019/10/15
Y1 - 2019/10/15
N2 - 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.
AB - 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.
KW - Disordered protein
KW - Docking interactions
KW - MAP kinases
KW - Mass spectrometry
KW - Peptide ligands
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U2 - 10.1073/pnas.1819533116
DO - 10.1073/pnas.1819533116
M3 - Article
C2 - 31578253
AN - SCOPUS:85073303953
SN - 0027-8424
VL - 116
SP - 21001
EP - 21011
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 42
ER -