scholarly journals In vitro reconstitution of Sgk3 activation by phosphatidylinositol-3-phosphate

2021 ◽  
Author(s):  
Daniel Pokorny ◽  
Linda Truebestein ◽  
Kaelin D Fleming ◽  
John E Burke ◽  
Thomas A Leonard
Keyword(s):  
Conformational Changes ◽  
Binding Pocket ◽  
Growth Factor Signaling ◽  
Allosteric Activation ◽  
Inhibitor Development ◽  
Allosteric Inhibitor ◽  
Px Domain ◽  
In Vitro Reconstitution ◽  
Phosphatidylinositol 3

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is activated by the phospholipid phosphatidylinositol-3-phosphate (PI3P) downstream of growth factor signaling and by Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers. Here, we show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation by PI3P. We demonstrate that PI3P binding induces large conformational changes in Sgk3 associated with its activation, and that the PI3P binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstituted Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to defining the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

scholarly journals Determinants of the Endosomal Localization of Sorting Nexin 1

2005 ◽  
Vol 16 (4) ◽  
pp. 2049-2057 ◽  
Author(s):  
Qi Zhong ◽  
Martin J. Watson ◽  
Cheri S. Lazar ◽  
Andrea M. Hounslow ◽  
Jonathan P. Waltho ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Early Endosome ◽  
Nmr Structure ◽  
Sorting Nexin ◽  
High Affinity ◽  
Terminal Domain ◽  
Px Domain ◽  
Phox Homology ◽  
Phosphatidylinositol 3

The sorting nexin (SNX) family of proteins is characterized by sequence-related phox homology (PX) domains. A minority of PX domains bind with high affinity to phosphatidylinositol 3-phosphate [PI(3)P], whereas the majority of PX domains exhibit low affinity that is insufficient to target them to vesicles. SNX1 is located on endosomes, but its low affinity PX domain fails to localize in vivo. The NMR structure of the PX domain of SNX1 reveals an overall fold that is similar to high-affinity PX domains. However, the phosphatidylinositol (PI) binding pocket of the SNX1 PX domain is incomplete; regions of the pocket that are well defined in high-affinity PX domains are highly mobile in SNX1. Some of this mobility is lost upon binding PI(3)P. The C-terminal domain of SNX1 is a long helical dimer that localizes to vesicles but not to the early endosome antigen-1–containing vesicles where endogenous SNX1 resides. Thus, the obligate dimerization of SNX1 that is driven by the C-terminal domain creates a high-affinity PI binding species that properly targets the holo protein to endosomes.

scholarly journals The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

2021 ◽  
Author(s):  
Krishna Chintaluri
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Ph Domain ◽  
Fyve Domain ◽  
Px Domain ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

scholarly journals In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0246319
Author(s):  
Raphael J. Eberle ◽  
Danilo S. Olivier ◽  
Carolina C. Pacca ◽  
Clarita M. S. Avilla ◽  
Mauricio L. Nogueira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Chikungunya Virus ◽  
In Vitro Study ◽  
3D Models ◽  
Binding Pocket ◽  
Starting Point ◽  
Low Cytotoxicity ◽  
Inhibitory Potential ◽  
Dynamics Simulations

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

scholarly journals Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

2002 ◽  
Vol 46 (6) ◽  
pp. 1805-1815 ◽  
Author(s):  
Jonathan Heddle ◽  
Anthony Maxwell
Keyword(s):  
Conformational Changes ◽  
Structural Data ◽  
Dna Gyrase ◽  
Binding Pocket ◽  
Quinolone Resistance ◽  
Resistance Mutations ◽  
Mutant Proteins ◽  
Close Proximity ◽  
To Come

ABSTRACT DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials. The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr122) where DNA is bound and cleaved. However, some quinolone resistance mutations are known to exist in GyrB. Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action. We have made and purified two such mutant proteins, GyrB(Asp426→Asn) and GyrB(Lys447→Glu), and characterized them in vitro. We found that the two proteins behave similarly to GyrA quinolone-resistant proteins. We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex. We suggest that the GyrB residues form part of a quinolone-binding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.

scholarly journals In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Inda Setyawati ◽  
Weronika K Stanek ◽  
Maria Majsnerowska ◽  
Lotteke J Y M Swier ◽  
Els Pardon ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Energy Coupling ◽  
Coupling Factor ◽  
In Vitro System ◽  
In Vitro Reconstitution ◽  
Reconstituted System ◽  
Kinetics Of

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.

scholarly journals The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

2021 ◽  
Author(s):  
Krishna Chintaluri
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Ph Domain ◽  
Fyve Domain ◽  
Px Domain ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

scholarly journals Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

2016 ◽  
Vol 113 (14) ◽  
pp. 3797-3802 ◽  
Author(s):  
Abrahim El Gamal ◽  
Vinayak Agarwal ◽  
Stefan Diethelm ◽  
Imran Rahman ◽  
Michelle A. Schorn ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Basis ◽  
Gene Locus ◽  
Bromine Atom ◽  
Larval Settlement ◽  
Binding Pocket ◽  
Carrier Protein ◽  
Substrate Binding Pocket ◽  
In Vitro Reconstitution

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

scholarly journals Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

2017 ◽  
Vol 214 (11) ◽  
pp. 3331-3346 ◽  
Author(s):  
Miguel Alejandro Lopez-Ramirez ◽  
Gregory Fonseca ◽  
Hussein A. Zeineddine ◽  
Romuald Girard ◽  
Thomas Moore ◽  
...  
Keyword(s):  
Cavernous Malformation ◽  
Angiogenesis Inhibitors ◽  
Cerebral Cavernous Malformations ◽  
Growth Factor Signaling ◽  
Vascular Endothelial ◽  
Micro Computed Tomography ◽  
Cavernous Malformations ◽  
In Vitro Reconstitution ◽  
Endothelial Growth Factor

KRIT1 mutations are the most common cause of cerebral cavernous malformation (CCM). Acute Krit1 gene inactivation in mouse brain microvascular endothelial cells (BMECs) changes expression of multiple genes involved in vascular development. These changes include suppression of Thbs1, which encodes thrombospondin1 (TSP1) and has been ascribed to KLF2- and KLF4-mediated repression of Thbs1. In vitro reconstitution of TSP1 with either full-length TSP1 or 3TSR, an anti-angiogenic TSP1 fragment, suppresses heightened vascular endothelial growth factor signaling and preserves BMEC tight junctions. Furthermore, administration of 3TSR prevents the development of lesions in a mouse model of CCM1 (Krit1ECKO) as judged by histology and quantitative micro-computed tomography. Conversely, reduced TSP1 expression contributes to the pathogenesis of CCM, because inactivation of one or two copies of Thbs1 exacerbated CCM formation. Thus, loss of Krit1 function disables an angiogenic checkpoint to enable CCM formation. These results suggest that 3TSR, or other angiogenesis inhibitors, can be repurposed for TSP1 replacement therapy for CCMs.

scholarly journals Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations

2021 ◽  
Vol 9 ◽  
Author(s):  
Kristen Park ◽  
Katelyn J. Hoff ◽  
Linnea Wethekam ◽  
Nicholas Stence ◽  
Margarita Saenz ◽  
...  
Keyword(s):  
Brain Development ◽  
Conformational Changes ◽  
Mechanistic Explanation ◽  
Binding Pocket ◽  
Missense Mutations ◽  
Tubulin Genes ◽  
Brain Malformations ◽  
The Impact ◽  
Β Tubulin

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

scholarly journals Reconstitution of Kinesin-1 Activation

2021 ◽  
Author(s):  
Kyoko Chiba ◽  
Kassandra M. Ori-McKenney ◽  
Shinsuke Niwa ◽  
Richard J. McKenney
Keyword(s):  
Conformational Changes ◽  
Regulatory Mechanism ◽  
Adaptor Proteins ◽  
Intramolecular Interactions ◽  
Motor Complex ◽  
In Vitro Reconstitution ◽  
Independent Mechanism ◽  
Microtubule Motor ◽  
Motor Interaction

AbstractAutoinhibition is an important regulatory mechanism for cytoskeletal motor proteins. Kinesin-1 (kinesin hereafter), the ubiquitous plus-end directed microtubule motor, is thought to be controlled by a complicated autoinihibition mechanism, but the molecular details remain unclear. Conformational changes mediated by intramolecular interactions between the C-terminal tail and N-terminal motor domains of the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The dimeric KHC also binds two copies of the kinesin light chains (KLCs), which have been implicated in both autoinhibition and cargo-dependent activation of the tetrameric motor complex, although the precise mechanisms remain opaque. Using in vitro reconstitution, we show that the KLC strongly inhibits the kinesin-microtubule interaction via an independent mechanism from the tail-motor interaction within KHC. Kinesin cargo-adaptor proteins that bind KLC activated processive movement of the kinesin tetramer but the landing rate of these activated complexes remained low. The addition of MAP7, which specifically binds to the KHC, strongly enhanced activated motor motility by dramatically increasing the landing rate and processivity of the activated kinesin motors. Our results support a model whereby the activity of the kinesin tetramer is regulated by independent tail- and KLC-based inhibition mechanisms, and that cargo-adaptor binding to the KLC directly releases both of these inhibitions. However, we find that a third component, a non-motor MAP is required for robust activity of the activated motor. Thus, human kinesin activity is regulated by a two-factor mechanism comprised of intramolecular allosteric regulation, as well as intermolecular kinesin-adaptor and kinesin-MAP interactions.

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