Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

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The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

Communications Biology ◽

10.1038/s42003-020-01459-2 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Meredith L. Jenkins ◽

Noah J. Harris ◽

Udit Dalwadi ◽

Kaelin D. Fleming ◽

Daniel S. Ziemianowicz ◽

...

Keyword(s):

Membrane Trafficking ◽

Rab Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Binding Interface ◽

Nucleotide Exchange Factor ◽

Biochemical Characterisation ◽

Hydrogen Deuterium ◽

Insight Into

AbstractThe TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

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Activation of GCN2 by the ribosomal P-stalk

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813352116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4946-4954 ◽

Cited By ~ 43

Author(s):

Alison J. Inglis ◽

Glenn R. Masson ◽

Sichen Shao ◽

Olga Perisic ◽

Stephen H. McLaughlin ◽

...

Keyword(s):

Conformational Changes ◽

Principal Component ◽

Protein Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Hdx Ms ◽

Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

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Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of β-arrestin 2 coupling

Scientific Reports ◽

10.1038/s41598-019-51016-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Kaushik Sarkar ◽

Lisa Joedicke ◽

Marta Westwood ◽

Rebecca Burnley ◽

Michael Wright ◽

...

Keyword(s):

Parathyroid Hormone ◽

Conformational Changes ◽

Transmembrane Domain ◽

Receptor Activation ◽

Parathyroid Hormone Receptor ◽

Exchange Mass Spectrometry ◽

Gpcr Activation ◽

Hydrogen Deuterium ◽

Binding Antibody ◽

Hdx Ms

Abstract Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

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Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

PeerJ ◽

10.7717/peerj.9408 ◽

2020 ◽

Vol 8 ◽

pp. e9408

Author(s):

Shanshan Wu ◽

Tam T.T.N. Nguyen ◽

Olga V. Moroz ◽

Johan P. Turkenburg ◽

Jens E. Nielsen ◽

...

Keyword(s):

Conformational Changes ◽

Solvent Accessibility ◽

Conformational Dynamics ◽

Structural Heterogeneity ◽

Catalytic Process ◽

Conformational Heterogeneity ◽

X Ray ◽

Exchange Mass Spectrometry ◽

Bacillus Lentus ◽

Hdx Ms

Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

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HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

10.1101/595553 ◽

2019 ◽

Author(s):

Zainab Ahdash ◽

Euan Pyle ◽

William J. Allen ◽

Robin A. Corey ◽

Ian Collinson ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Protein Transport ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Conformational Dynamics ◽

Brownian Ratchet ◽

Exchange Mass Spectrometry ◽

Dependent Transport ◽

Hdx Ms

AbstractThe bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel – SecYEG – associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

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Rab GTPases: master regulators that establish the secretory and endocytic pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e16-10-0737 ◽

2017 ◽

Vol 28 (6) ◽

pp. 712-715 ◽

Cited By ~ 113

Author(s):

Suzanne R. Pfeffer

Keyword(s):

Membrane Trafficking ◽

Membrane Microdomains ◽

Rab Gtpase ◽

Rab Proteins ◽

Rab Gtpases ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Open Questions ◽

Endocytic Pathways

Several of the most important discoveries in the field of membrane traffic have come from studies of Rab GTPases by Marino Zerial and Peter Novick and their colleagues. Zerial was the first to discover that Rab GTPases represent identity markers for different membrane-bound compartments, and each Rab organizes a collection of specific effectors into function-specifying membrane microdomains to carry out receptor trafficking. Novick discovered that the order (and thus polarity) of Rab GTPases along the secretory and endocytic pathways are established by their specific, cognate guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which partner with one Rab to regulate the subsequent- and prior-acting Rabs. Such so-called Rab cascades have evolved to establish domains that contain unique Rab proteins and their cognate effectors, which drive all steps of membrane trafficking. These findings deserve much broader recognition by the biomedical research community and are highlighted here, along with open questions that require serious attention for full understanding of the molecular basis of Rab GTPase-regulated membrane trafficking in eukaryotic cells.

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Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase fromEntamoeba histolytica

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14011388 ◽

2014 ◽

Vol 70 (7) ◽

pp. 933-937 ◽

Cited By ~ 3

Author(s):

Vijay Kumar Srivastava ◽

Mintu Chandra ◽

Sunando Datta

Keyword(s):

Entamoeba Histolytica ◽

Conformational Changes ◽

Membrane Trafficking ◽

Vital Role ◽

Diffusion Method ◽

Monoclinic Space Group ◽

Rab Gtpases ◽

Data Set ◽

Cell Parameters ◽

Ras Superfamily

Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasiteEntamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes.Entamoeba histolyticaRabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8–16% sequence identity with other GTPases. Recent studies suggested thatEhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action ofEhRabX3. To provide structural insights into the inclusive action of this unique GTPase,EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single nativeEhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space groupC2, with unit-cell parametersa= 198.6,b= 119.3,c= 89.2 Å, β = 103.1°. Preliminary analysis of the data using theMatthews Probability Calculatorsuggested the presence of four to six molecules in the asymmetric unit.

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Structural Clues to Rab GTPase Functional Diversity

Journal of Biological Chemistry ◽

10.1074/jbc.r500003200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 15485-15488 ◽

Cited By ~ 114

Author(s):

Suzanne R. Pfeffer

Keyword(s):

Structural Analysis ◽

Functional Diversity ◽

Membrane Trafficking ◽

Crystallographic Analysis ◽

Protein Family ◽

Rab Gtpase ◽

Rab Gtpases ◽

Important Protein ◽

Partner Proteins ◽

Insight Into

Rab GTPases are key regulators of membrane trafficking in eukaryotes. Recent structural analysis of a number of Rabs, either alone or in complex with partner proteins, has provided new insight into the importance of both conserved and non-conserved features of these proteins that specify their unique functions and localizations. This review will highlight what we have learned from crystallographic analysis of this important protein family.

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HDX-MS reveals nucleotide-dependent, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

eLife ◽

10.7554/elife.47402 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Zainab Ahdash ◽

Euan Pyle ◽

William John Allen ◽

Robin A Corey ◽

Ian Collinson ◽

...

Keyword(s):

Conformational Changes ◽

Opposite Effect ◽

Protein Transport ◽

Protein Translocation ◽

Brownian Ratchet ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Dependent Transport ◽

Hdx Ms

The bacterial Sec translocon is a multi-protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel – SecYEG – associates with the motor protein SecA to mediate the ATP-dependent transport of pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Nature Communications ◽

10.1038/s41467-020-20032-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ruyu Jia ◽

Chloe Martens ◽

Mrinal Shekhar ◽

Shashank Pant ◽

Grant A. Pellowe ◽

...

Keyword(s):

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Conformational Dynamics ◽

Md Simulations ◽

Allosteric Coupling ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

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