Publications by authors named "David Hamelin"

8 Publications

  • Page 1 of 1

Probing Protein-Membrane Interactions and Dynamics Using Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Methods Mol Biol 2021 ;2263:465-485

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.

Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to these processes being accomplished appropriately is the highly controlled recruitment and assembly of proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein-membrane interactions can be facilitated by utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral membrane proteins upon association with membrane. Particular reference will be made to the analysis of the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP)-containing membranes to illustrate specific methodological principles.
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http://dx.doi.org/10.1007/978-1-0716-1197-5_22DOI Listing
June 2021

The juxtamembrane linker in neutral sphingomyelinase-2 functions as an intramolecular allosteric switch that activates the enzyme.

J Biol Chem 2019 05 19;294(18):7488-7502. Epub 2019 Mar 19.

From the Departments of Biochemistry and Cell Biology,

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.
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http://dx.doi.org/10.1074/jbc.RA118.007288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509483PMC
May 2019

A single discrete Rab5-binding site in phosphoinositide 3-kinase β is required for tumor cell invasion.

J Biol Chem 2019 03 18;294(12):4621-4633. Epub 2019 Jan 18.

From the Departments of Molecular Pharmacology and

Phosphoinositide 3-kinase β (PI3Kβ) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln and Ile) in the helical domain of the catalytic subunit (p110β) of PI3Kβ whose mutation disrupts binding to Rab5. To better define the Rab5-p110β interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an pulldown assay with GST-Rab5 Of the 35 p110β helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gβγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110β as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln and Ile residues. Analysis of the Rab5-PI3Kβ interaction by hydrogen-deuterium exchange MS identified p110β peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110β or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110β had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kβ activity , the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110β helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kβ coupling .
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http://dx.doi.org/10.1074/jbc.RA118.006032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433078PMC
March 2019

Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.

J Med Chem 2018 12 28;61(23):10463-10472. Epub 2018 Nov 28.

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography , University of California, San Diego , La Jolla , California 92093 , United States.

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688905PMC
December 2018

Structural and mechanistic insights into the function of the unconventional class XIV myosin MyoA from .

Proc Natl Acad Sci U S A 2018 11 22;115(45):E10548-E10555. Epub 2018 Oct 22.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada;

Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of and MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.
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http://dx.doi.org/10.1073/pnas.1811167115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233092PMC
November 2018

Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5.

Nat Commun 2018 09 14;9(1):3772. Epub 2018 Sep 14.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.

The GTPase Rab11 plays key roles in receptor recycling, oogenesis, autophagosome formation, and ciliogenesis. However, investigating Rab11 regulation has been hindered by limited molecular detail describing activation by cognate guanine nucleotide exchange factors (GEFs). Here, we present the structure of Rab11 bound to the GEF SH3BP5, along with detailed characterization of Rab-GEF specificity. The structure of SH3BP5 shows a coiled-coil architecture that mediates exchange through a unique Rab-GEF interaction. Furthermore, it reveals a rearrangement of the switch I region of Rab11 compared with solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutation of switch I provides insights into the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases present on Rab11-positive organelles. Moreover, we show that GEF-deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. Overall, our results give molecular insight into Rab11 regulation, and how Rab-GEF specificity is achieved.
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http://dx.doi.org/10.1038/s41467-018-06196-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138693PMC
September 2018

Probing the Architecture, Dynamics, and Inhibition of the PI4KIIIα/TTC7/FAM126 Complex.

J Mol Biol 2018 09 18;430(18 Pt B):3129-3142. Epub 2018 Jul 18.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2. Electronic address:

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the lipid kinase primarily responsible for generating the lipid phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, which acts as the substrate for generation of the signaling lipids PIP and PIP. PI4KIIIα forms a large heterotrimeric complex with two regulatory partners, TTC7 and FAM126. We describe using an integrated electron microscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) approach to probe the architecture and dynamics of the complex of PI4KIIIα/TTC7/FAM126. HDX-MS reveals that the majority of the PI4KIIIα sequence was protected from exchange in short deuterium pulse experiments, suggesting presence of secondary structure, even in putative unstructured regions. Negative stain electron microscopy reveals the shape and architecture of the full-length complex, revealing an overall dimer of PI4KIIIα/TTC7/FAM126 trimers. HDX-MS reveals conformational changes in the TTC7/FAM126 complex upon binding PI4KIIIα, including both at the direct TTC7-PI4KIIIα interface and at the putative membrane binding surface. Finally, HDX-MS experiments of PI4KIIIα bound to the highly potent and selective inhibitor GSK-A1 compared to that bound to the non-specific inhibitor PIK93 revealed substantial conformational changes throughout an extended region of the kinase domain. Many of these changes were distant from the putative inhibitor binding site, showing a large degree of allosteric conformational changes that occur upon inhibitor binding. Overall, our results reveal novel insight into the regulation of PI4KIIIα by its regulatory proteins TTC7/FAM126, as well as additional dynamic information on how selective inhibition of PI4KIIIα is achieved.
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http://dx.doi.org/10.1016/j.jmb.2018.07.020DOI Listing
September 2018

Conformational sampling of membranes by Akt controls its activation and inactivation.

Proc Natl Acad Sci U S A 2018 04 9;115(17):E3940-E3949. Epub 2018 Apr 9.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, 1030 Vienna, Austria;

The protein kinase Akt controls myriad signaling processes in cells, ranging from growth and proliferation to differentiation and metabolism. Akt is activated by a combination of binding to the lipid second messenger PI(3,4,5)P and its subsequent phosphorylation by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2. The relative contributions of these mechanisms to Akt activity and signaling have hitherto not been understood. Here, we show that phosphorylation and activation by membrane binding are mutually interdependent. Moreover, the converse is also true: Akt is more rapidly dephosphorylated in the absence of PIP, an autoinhibitory process driven by the interaction of its PH and kinase domains. We present biophysical evidence for the conformational changes in Akt that accompany its activation on membranes, show that Akt is robustly autoinhibited in the absence of PIP irrespective of its phosphorylation, and map the autoinhibitory PH-kinase interface. Finally, we present a model for the activation and inactivation of Akt by an ordered series of membrane binding, phosphorylation, dissociation, and dephosphorylation events.
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http://dx.doi.org/10.1073/pnas.1716109115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924885PMC
April 2018