Dataset Information

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

ABSTRACT: The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches.

SUBMITTER: Fowler ML

PROVIDER: S-EPMC4832280 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

Fowler Melissa L ML McPhail Jacob A JA Jenkins Meredith L ML Masson Glenn R GR Rutaganira Florentine U FU Shokat Kevan M KM Williams Roger L RL Burke John E JE

Protein science : a publication of the Protein Society 20160201 4

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidyl ...[more]

PMID: 26756197

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Project description:Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique to interrogate protein structure and dynamics. With the ability to study almost any protein without a size limit, including intrinsically disordered ones, HDX-MS has shown fast growing importance as a complement to structural elucidation techniques. Current experiments compare two or more related conditions (sequences, interaction partners, excipients, conformational states, etc.) to determine statistically significant differences at a number of fixed time points and highlight areas of changed structural dynamics in the protein. The work presented here builds on the fundamental research performed in the early days of the technique and re-examines exchange rate calculations with the aim of establishing HDX-MS as an absolute and quantitative, rather than relative and qualitative, measurement. We performed millisecond HDX-MS experiments on a mixture of three unstructured peptides (angiotensin, bradykinin, and atrial natriuretic peptide amide rat) and compared experimental deuterium uptake curves with theoretical ones predicted using established exchange rate calculations. With poly-dl-alanine (PDLA) commonly used as a reference,, we find that experimental rates are sometimes faster than theoretically possible, while they agree much better, and are never faster, with the fully unstructured trialanine peptide (3-Ala). Molecular dynamics (MD) simulations confirm the high helical propensity of the longer and partially structured PDLA peptides, which need as few as 15 residues to form a stable helix and are therefore not suitable as an unstructured reference. Reanalysis of previously published data by Weis et al. at 100 mM NaCl however still shows a discrepancy with predictions based on 3-Ala in the absence of salt, highlighting the need for a better understanding of salt effects on exchange rates. Such currently unquantifiable salt effects prevent us from proposing a comprehensive, universal calibration framework at the moment. Nevertheless, an accurate recalibration of intrinsic exchange rate calculations is crucial to enable kinetic modeling of the exchange process and to ultimately allow HDX-MS to move toward a direct link with atomistic structural models.

Dataset Information

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

Publications

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

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