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HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone.

ABSTRACT: 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 ?-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent ?-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.

SUBMITTER: Kielkopf CS

PROVIDER: S-EPMC6319766 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone.

Kielkopf Claudia S CS Ghosh Madhubrata M Anand Ganesh S GS Brown Simon H J SHJ

Protein science : a publication of the Protein Society 20181220 2

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) re ...[more]

PMID: 30353968

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Project description:BackgroundCalmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR.ResultsWe provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified.ConclusionOur results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Dataset Information

HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone.

Publications

HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone.

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