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An Affinity-Based, Cysteine-Specific ATP Analog for Kinase-Catalyzed Crosslinking.

ABSTRACT: Kinases mediate cell signaling pathways by catalyzing protein phosphorylation. Irregularities in kinase activity are directly associated with disease conditions. Therefore, methods to identify substrates of a particular kinase are needed to understand signaling cascades in normal and diseased states. Photocrosslinking ATP analogs provide powerful tools to study kinases by covalently linking kinases with substrates. However, the involvement of UV light and nonspecific reactivity of current ATP-photocrosslinkers challenge kinase-substrate identification. We report here an affinity-based crosslinking ATP analog, ATP-methylacrylamide (ATP-MAc), that contains a cysteine-reactive acrylamide crosslinking group, which avoids the UV irradiation and non-specific reactivity of prior analogs. Using in vitro kinase assays, ATP-MAc acts as a kinase co-substrate and covalently crosslinks only kinases containing cysteines in the active site. ATP-MAc was also able to crosslink cellular proteins in lysates, documenting compatibility with cell-based studies.

SUBMITTER: Fouda AE

PROVIDER: S-EPMC8084585 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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An Affinity-Based, Cysteine-Specific ATP Analog for Kinase-Catalyzed Crosslinking.

Fouda Ahmed E AE Gamage Aparni K AK Pflum Mary Kay H MKH

Angewandte Chemie (International ed. in English) 20210323 18

Kinases mediate cell signaling pathways by catalyzing protein phosphorylation. Irregularities in kinase activity are directly associated with disease conditions. Therefore, methods to identify substrates of a particular kinase are needed to understand signaling cascades in normal and diseased states. Photocrosslinking ATP analogs provide powerful tools to study kinases by covalently linking kinases with substrates. However, the involvement of UV light and nonspecific reactivity of current ATP-ph ...[more]

PMID: 33619842

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Project description:DEAD-box helicases are important regulators of biomolecular condensates. However, the mechanisms through which these enzymes affect the dynamics of biomolecular condensates have not been systematically explored. Here, we demonstrate the mechanism by which mutation of a DEAD-box helicaseâ€™s catalytic core alters ribonucleoprotein condensate dynamics in the presence of ATP. Through altering RNA length within the system, we are able to attribute the altered biomolecular dynamics and material properties to physical crosslinking of RNA facilitated by the mutant helicase. These results suggest the mutant condensates approach a gel transition when RNA length is increased to lengths comparable to eukaryotic mRNA. Lastly, we show that this crosslinking effect is tunable with ATP concentration, uncovering a system whose RNA mobility and material properties vary with enzyme activity. More generally, these findings point to a fundamental mechanism for modulating condensate dynamics and emergent material properties through nonequilibrium, molecular-scale interactions.SignificanceBiomolecular condensates are membraneless organelles which organize cellular biochemistry. These structures have a diversity of material properties and dynamics which are crucial to their function. How condensate properties are determined by biomolecular interactions and enzyme activity remain open questions. DEAD-box helicases have been identified as central regulators of many protein-RNA condensates, though their specific mechanistic roles are ill-defined. In this work, we demonstrate that a DEAD-box helicase mutation crosslinks condensate RNA in an ATP-dependent fashion via protein-RNA clamping. Protein and RNA diffusion can be tuned with ATP concentration, corresponding to an order of magnitude change in condensate viscosity. These findings expand our understanding of control points for cellular biomolecular condensates that have implications for medicine and bioengineering.

Dataset Information

An Affinity-Based, Cysteine-Specific ATP Analog for Kinase-Catalyzed Crosslinking.

Publications

An Affinity-Based, Cysteine-Specific ATP Analog for Kinase-Catalyzed Crosslinking.

Similar Datasets

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