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

Fowler, M.L.; McPhail, Jacob A.; Jenkins, Meredith L.; Masson, Glenn R.; Rutaganira, Florentine U.; Shokat, Kevan M.; Williams, Roger L.; Burke, John E.

doi:10.1002/pro.2879

Cited by 42 publications

(41 citation statements)

References 54 publications

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“…This structure revealed the molecular basis of its interaction with Rab11, as well as revealing the binding of compound 1 to PI4KIIIβ, however, this construct only crystallized in the presence of compound 1 . To generate crystals of PI4KIIIβ bound to compound 9 we used a novel crystallization construct generated through the use of a hydrogen deuterium exchange mass spectrometry based approach 21 . This construct allowed us to generate crystals of PI4KIIIβ bound to GDP loaded Rab11 in the absence of inhibitors.…”

Section: Resultsmentioning

confidence: 99%

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ

et al. 2016

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Type III Phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking, and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles, and may lead to novel anti-viral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design we have designed novel potent and selective (>1000 fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed anti-viral activity against Hepatitis C Virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as anti-viral therapeutics.

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Section: Resultsmentioning

confidence: 99%

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ

et al. 2016

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“…This region had no protection from amide exchange in the apo state, revealing it to be disordered, with a very strong stabilization (> 80% decrease in exchange) in the presence of c10orf76, indicating a disorder-to-order transition upon c10orf76 binding ( Fig 1H). This N-lobe kinase linker is dispensable for lipid kinase activity, as it can be removed with a minimal effect on PI4KB catalytic activity [19]. In addition to this change, there were multiple smaller decreases in exchange in the helical domain (131-138, 149-157, 159-164, and 183-204) and kinase domain (676-688, 725-734, and 738-765).…”

Section: Hdx-ms Reveals That Pi4kb and C10orf76 Form An Extended Intementioning

confidence: 99%

“…PI4KB is a multi-domain lipid kinase containing a disordered N-terminus, a helical domain, and a bi-lobal kinase domain [14,19]. PI4KB is a multi-domain lipid kinase containing a disordered N-terminus, a helical domain, and a bi-lobal kinase domain [14,19].…”

Section: Introductionmentioning

confidence: 99%

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

et al. 2019

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The lipid kinase PI4KB, which generates phosphatidylinositol 4phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogendeuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complexdisrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

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“…While HDX can also be probed by NMR with residue level resolution, this approach is generally restricted to small soluble proteins that are amenable to solution state NMR analysis . By contrast, mass spectrometers have no inherent size limit and have been used to study systems as large and complex as intact ribosomal complexes . HDX‐MS experiments require submicromolar concentrations of sample and can, under certain circumstances, provide residue level information …”

Section: Introductionmentioning

confidence: 99%

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

2019

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Much of our understanding of protein structure and mechanistic function has been derived from static high‐resolution structures. As structural biology has continued to evolve it has become clear that high‐resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX‐MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX‐MS to monitor these dynamic changes in proteins. We examine how HDX‐MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. Statement for a Broad Audience The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

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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

Cited by 42 publications

References 54 publications

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

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