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An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

ABSTRACT: Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(?185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle x-ray scattering and isotope-assisted chemical cross-linking to apoA-I(?185-243) in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of high density lipoprotein biogenesis.

SUBMITTER: Melchior JT

PROVIDER: S-EPMC4777873 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

Melchior John T JT Walker Ryan G RG Morris Jamie J Jones Martin K MK Segrest Jere P JP Lima Diogo B DB Carvalho Paulo C PC Gozzo Fábio C FC Castleberry Mark M Thompson Thomas B TB Davidson W Sean WS

The Journal of biological chemistry 20160111 10

Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(Δ185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existin ...[more]

PMID: 26755744

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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 ?-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.

Dataset Information

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

Publications

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

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