Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers

Krist, David T.; Statsyuk, Alexander V.

doi:10.1021/acs.biochem.5b00625

Cited by 9 publications

(7 citation statements)

References 19 publications

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“…they are selective for side-chain primary amines or sulfhydryls, which are not necessarily close to relevant binding surfaces. Recently, however, new crosslinking reagents that are highly reactive and relatively nonspecific have proven useful in proteinprotein interaction studies and show promise for protein structure mapping (17,18).…”

Section: Significancementioning

confidence: 99%

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

Parker

Goncz

Krist

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Significancementioning

confidence: 99%

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

Parker

Goncz

Krist

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Moreover, amine-specific linkers with different reactive groups and spacer distances were utilized to dissect the flexible or disordered interfaces of macromolecular complexes such as MICAL3, ELKS, and Rab8A; SLAIN2, CLASP2, and ch-TOG (Liu et al, 2017); NLRP1 and DPP9 (Hollingsworth et al, 2021); Rpn2 and Rpn13 (Gong et al, 2020). Recent developments in linker chemistry have produced crosslinker reagents that are cleavable, photo-activatable, or heterobifunctional, permitting controlled crosslink reactions, low background, improved sensitivity (Beard et al, 2021;Krist and Statsyuk 2015), and even in a native cellular environment (Xie et al, 2021). For instance, a crosslinker bearing a photoreactive moiety as well as a reducible spacer have been developed.…”

Section: Interfaces Revealed By a Chemical Crosslinker Coupled To Msmentioning

confidence: 99%

Unveiling the biological interface of protein complexes by mass spectrometry-coupled methods

Lim

2022

Preprint

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Most biomolecules become functional and bioactive by forming protein complexes through interaction with ligands that are diverse in size, shape, and physicochemical properties. In the complex biological milieu, the interaction is ligand-specific, driven by molecular sensing and recognition of a binding interface localized within a protein structure. Mapping interfaces of protein complexes is a highly sought area of research as it delivers fundamental insights into proteomes and pathology and hence strategies for therapeutics. While X-ray crystallography and electron microscopy still serve as a gold standard for structural elucidation of protein complexes, artificial and static analytic nature thereof often results in a non-native interface that otherwise might be negligible or non-existent in biological environment. In recent years, the mass spectrometry-coupled approaches, chemical crosslinking (CLMS) and hydrogen-deuterium exchange (HDMS), have become valuable analytic complements to traditional techniques. These methods explicitly identify hot residues and motifs embedded in binding interfaces, in particular, for which the interaction is predominantly dynamic, transient, and/or caused by an intrinsically disordered domain. Here we review the principal role of CLMS and HDMS in protein structural biology with a particular emphasis on the contribution of recent examples to exploring biological interfaces. In addition, we describe recent studies that utilized these methods to expand our understanding of protein complex formation and related biological processes and to increase probability of structure-based drug design.

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“…The utility of photocrosslinker incorporation into E2s for E3 study has also been demonstrated. Crosslinker incorporation by chemical labeling has enabled the mapping of Cys E3 catalytic residues (Krist and Statsyuk, 2015) and, more recently, incorporation into an E2 by chemical synthesis has been used to generate a photoaffinity probe for a SUMO E3 (Zhang et al, 2019). However, the utility of these technologies as ABPs has not been demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases

Mathur

Fletcher

Branigan

et al. 2020

Cell Chemical Biology

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Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers

Cited by 9 publications

References 19 publications

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

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

Unveiling the biological interface of protein complexes by mass spectrometry-coupled methods

Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases

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