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Transient exposure of a buried phosphorylation site in an autoinhibited protein.

ABSTRACT: Autoinhibition is a mechanism used to regulate protein function, often by making functional sites inaccessible through the interaction with a cis-acting inhibitory domain. Such autoinhibitory domains often display a substantial degree of structural disorder when unbound, and only become structured in the inhibited state. These conformational dynamics make it difficult to study the structural origin of regulation, including effects of regulatory post-translational modifications. Here, we study the autoinhibition of the Dbl Homology domain in the protein Vav1 by the so-called acidic inhibitory domain. We use molecular simulations to study the process by which a mostly unstructured inhibitory domain folds upon binding and how transient exposure of a key buried tyrosine residue makes it accessible for phosphorylation. We show that the inhibitory domain, which forms a helix in the bound and inhibited stated, samples helical structures already before binding and that binding occurs via a molten-globule-like intermediate state. Together, our results shed light on key interactions that enable the inhibitory domain to sample a finely tuned equilibrium between an inhibited and a kinase-accessible state.

SUBMITTER: Orioli S

PROVIDER: S-EPMC8758417 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Transient exposure of a buried phosphorylation site in an autoinhibited protein.

Orioli Simone S Henning Hansen Carl G CG Lindorff-Larsen Kresten K

Biophysical journal 20211203 1

Autoinhibition is a mechanism used to regulate protein function, often by making functional sites inaccessible through the interaction with a cis-acting inhibitory domain. Such autoinhibitory domains often display a substantial degree of structural disorder when unbound, and only become structured in the inhibited state. These conformational dynamics make it difficult to study the structural origin of regulation, including effects of regulatory post-translational modifications. Here, we study th ...[more]

PMID: 34864046

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Project description:Non-covalent protein-protein interactions are one of the most fundamental building blocks in cellular signalling pathways. Despite this, they have been historically hard to identify using conventional methods due to their often weak and transient nature. Using genetic code expansion and incorporation of commercially available unnatural amino acids, we have developed a highly accessible method whereby interactions between biotinylated ubiquitin-like protein (UBL) probes and their binding partners can be stabilised using ultraviolet (UV) light-induced crosslinks. The stabilised protein complexes can be purified using affinity purification and identified by mass spectrometry. The resultant covalent bonds can withstand even the harshest washing conditions, allowing for the removal of indirect binders whilst retaining and capturing weak and transient interactors that are commonly lost during wash steps. This technique is widely applicable and highly effective for identifying site-selective non-covalent interactors. Members of our team have previously demonstrated the benefit of this method using the small ubiquitin-like modifier (SUMO). Here, we provide further proof-of-principle validation of the method and highlight its generality by applying an optimised workflow to a lesser studied UBL, interferon stimulated gene 15 (ISG15). We show that this method is able to capture known ISG15 interactors from a complex protein mixture in a site-selective manner, only capturing proteins that specifically interact with the region of ISG15 where the unnatural amino acid was incorporated. This exquisite degree of sensitivity and specificity greatly improves upon previous screens aimed at identifying downstream non-covalent binders, or readers, of ISG15. Taken together, the approach opens the possibility of characterising previously undetected protein-protein interactions, with the potential of elucidating molecular mechanisms behind the most complex and poorly understood processes in the cell.

Dataset Information

Transient exposure of a buried phosphorylation site in an autoinhibited protein.

Publications

Transient exposure of a buried phosphorylation site in an autoinhibited protein.

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