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Dynamics of protein kinases and pseudokinases by HDX-MS.

ABSTRACT: Dynamics of the protein kinase fold are deeply intertwined with its structure. The past three decades of kinase biophysical studies revealed key dynamic features of the kinase domain and, more recently, how these features may endow catalytically impaired kinases-or pseudokinases-with signaling properties. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is proving to be a valuable approach for studies of kinase and pseudokinase domain dynamics. Here, we briefly discuss the methods that have provided insights into protein kinase dynamics, describe how HDX-MS is being used to answer questions in the kinase/pseudokinase field, and provide a detailed protocol for collecting an HDX-MS dataset to study the impacts of small molecule binding to a pseudokinase domain. As more small molecules are discovered that can disrupt pseudokinase conformations, HDX-MS is likely to be a powerful approach for exploring drug-induced changes in pseudokinase dynamics and structure.

SUBMITTER: Sheetz JB

PROVIDER: S-EPMC9148214 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Dynamics of protein kinases and pseudokinases by HDX-MS.

Sheetz Joshua B JB Lemmon Mark A MA Tsutsui Yuko Y

Methods in enzymology 20220408

Dynamics of the protein kinase fold are deeply intertwined with its structure. The past three decades of kinase biophysical studies revealed key dynamic features of the kinase domain and, more recently, how these features may endow catalytically impaired kinases-or pseudokinases-with signaling properties. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is proving to be a valuable approach for studies of kinase and pseudokinase domain dynamics. Here, we briefly discuss the met ...[more]

PMID: 35525545

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Project description:Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded β-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.

Dataset Information

Dynamics of protein kinases and pseudokinases by HDX-MS.

Publications

Dynamics of protein kinases and pseudokinases by HDX-MS.

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