cdna of rab8a  (Addgene inc)

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Structure of pRab8a in Complex with the Phospho-Rab Binding Domain of RILPL2 (A) Heterotetrameric assembly of two pRab8a molecules bridged by a central α-helical dimer of the phospho-Rab binding domain of RILPL2 (129–165). The two chains of RILPL2 are in magenta and dark yellow. For pRab8a, switch 1 is shown in blue, switch 2 in red. (B) View of the complex down the 2-fold axis of the heterotetramer, 90° relative to orientation in (A). (C) Stick model of the RH2 domain of RILPL2. Rabs are stripped from the complex in this view, except for short segments of switch 1 and switch 2 (gray sticks). (D) Domain organization of RILPL1/2 showing the RH domains and their interacting partners. The sequence corresponds to RILPL2. (E) Simplified representation of the Rab:RILPL2 interface showing contacts between one molecule of Rab8a and the dimer of RILPL2. Polar interactions are indicated in dotted blue lines. Switch 1 (Sw1) and switch 2 are indicated. (F) Isothermal titration calorimetry analyses of the interactions between pRab8a and the phospho-Rab binding domain of RILPL2. Left, titration of RILPL2 (residues 129–165) into pRab8a(GTP). Right, titration of RILPL2 into Rab8a(GTP).

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Binding Assay, Sequencing, Isothermal Titration Calorimetry, Titration

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Mutational Analyses Reveal Hotspots of pRab8a:RILPL2 Interactions (A) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G], HA-Rab8a and WT or mutant RILPL2-GFP. At 48 h post transfection, cells were treated with ±500 nM MLi-2 for 90 min and then lysed. Upper panel, labeled IP:GFP: RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx western blot imaging system with the indicated antibodies at 0.5–1 μg/mL concentration. Lower panel, labeled input: 10 μg whole-cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments. (B) Same as A, but HEK293 cells were transiently transfected with WT or mutant HA-Rab8a as well as Flag-LRRK2[R1441G] and RILPL2-GFP WT. At 48 h post transfection, cells were treated with ±500 nM MLi-2 for 90 min and then lysed. RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and as in (A), immunoprecipitates and input were evaluated by immunoblotting with the indicated antibodies. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments. (C) Sequence alignment of the first α helix (α1) of the RILP family RH2 domains. Residues corresponding to the second α helix (α2) of RILP are not shown. Red circles are hotspots for the interactions where mutations severely reduce affinity between pRab8a and RILPL2. Blue circles indicate residues that are tolerant to mutations. The α-helical secondary structure above the alignment corresponds to RILPL2.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Transfection, Construct, Expressing, Mutagenesis, Labeling, Immunoprecipitation, Western Blot, Imaging, Concentration Assay, Sequencing

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Evidence that RILPL2 Binds to the GTP Bound Conformation of Phosphorylated Rab8a in Cells (A) Direct in vitro pull-downs were performed using purified His 6 -tagged RILPL2 (full length) as bait and untagged Rab8a as prey. Rab8a species were either non-phosphorylated (Rab8a) or phosphorylated (pRab8a). The GTP forms were stabilized via the Q67L mutation in switch 2. The GDP form of Rab8a was prepared by in vitro exchange using wild-type (WT) Rab8a before the phosphorylation reaction to generate pRab8a(GDP). Protein concentrations were 10 μM for bait and prey, inputs are 2 μg; n ≥ 3, Coomassie stain for visualization. Dotted lines emphasize that only pRab8a(GTP) binds to RILPL2. (B) HEK293 cells were transiently transfected with constructs expressing the indicated components. 24 h post transfection, cells were treated with ±100 nM MLi-2 for 90 min and then lysed. Upper panel, labeled IP:GFP: RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx western blot imaging system with the indicated antibodies at 0.5–1 μg/mL concentration. Lower panel, labeled input: 10 μg whole-cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: In Vitro, Purification, Mutagenesis, Phospho-proteomics, Staining, Transfection, Construct, Expressing, Labeling, Immunoprecipitation, Western Blot, Imaging, Concentration Assay

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: MyoVa Interactions with the RH1 Domain Enhance the Affinity of RILPL2 to pRab8a (A) Pull-downs of (p)Rab8a and RILPL2 in the presence of MyoVa(GTD). Input proteins are in the upper left panel, while duplicate pull-downs are shown to the right. Phosphorylated Rab8a (pRab8a) is highlighted in the pull-down lanes with red (+) labels. Bait and prey proteins were used at 2.5 μM. (B) Control experiment showing that no interactions are observed between His 6 -tagged MyoVa and pRab8a/Rab8a. (C) Quantification of densitometry readings of pRab8a pull-downs from three independent experiments (p < 0.005). (D) Modeling of full-length RILPL2 using ribbons and electrostatic surfaces. The RH1 domain of mouse RILPL2 was connected to the RH2 domain of human RILPL2. Residue numbers correspond to the human protein.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Control, Residue

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Structural Comparisons of Rab:Effector Complexes (A) Structure of Rab7 in complex with the Rab binding domain of RILP. (B) Superposition of RILPL2 onto a single binding interface of Rab7:RILP, showing conservation of the α-helical coiled coil. The figure is rotated 90° along the horizontal axis, relative to (A). (C) Structure of Rab8a in complex with OCRL1. The dashed circle denotes the region that sterically clashes with pT72 of pRab8a. (D) Close-up view of the switch 2 region denoted by the dashed circle. Here, pRab8a (red) from the complex with RILPL2 is superimposed onto the structure of Rab8a (gray) in complex with OCRL1. The distances between the methyl groups from the β-branched sidechains of pT72 and Ile71 are shown to highlight the steric clashes.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Binding Assay

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Model for the Control of Rab8a Functions by LRRK2 Rab29 recruits LRRK2 to membranes and Rab8a is subsequently phosphorylated by LRRK2. RILPL2 is then recruited to membranes by pRab8a via the X-cap. RILPL2 is an adaptor that links pRab8a to the GTD of MyoVa. The structure of the mouse complex of RILPL2 with myosin was used to generate this figure (PDB: 4kp3 ; Wei et al., 2013 ).

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Control

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet:

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Recombinant, Residue, Mutagenesis, Software

Journal: bioRxiv

Article Title: Structural basis for Rab8a GTPase recruitment of RILPL2 via LRRK2 phosphorylation of switch 2

doi: 10.1101/739813

Figure Lengend Snippet: A , heterotetrameric assembly of two pRab8a molecules bridged by a central α-helical dimer of the phospho-Rab binding domain of RILPL2 (129-165). The two chains of RILPL2 are in magenta and dark yellow. For pRab8a, switch 1 is shown in blue, switch 2 in red. B , view of the complex down the two-fold axis of the heterotetramer, 90° relative to orientation in A. C , cartoon representation of the contacts between one molecule of Rab8a and the dimer of RILPL2. Polar interactions are indicated in dotted blue lines. D , isothermal titration calorimetry analyses of the interactions between pRab8a and phospho-Rab binding domain of RILPL2. Left , titration of RILPL2 (residues 129-165) into pRab8a(GTP). Right , titration of RILPL2 into Rab8a(GTP).

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E.coli expression.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Titration

Journal: bioRxiv

Article Title: Structural basis for Rab8a GTPase recruitment of RILPL2 via LRRK2 phosphorylation of switch 2

doi: 10.1101/739813

Figure Lengend Snippet: A , view of an interface between Rab8a and the dimer of RILPL2. B , stick model of the interactions at the X-cap of RILPL2. C , electron density (2Fo-Fc, 1.2σ) at the site of pT72 (chain B) binding to R132 RL2 (chain D). The side chain of L135 RL2 from chain C of RILPL2 lies within van der Waals contact (4 Å) of the β-branched methyl group of pT72. D , electrostatic surface rendering of the X-cap. Blue is positive and red is negative, while switch 1 and 2 of pRab8a are ribbons with key residues represented as sticks.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E.coli expression.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural basis for Rab8a GTPase recruitment of RILPL2 via LRRK2 phosphorylation of switch 2

doi: 10.1101/739813

Figure Lengend Snippet: A , Direct in vitro pulldowns were performed using purified His 6 -tagged RILPL2 ( full length ) as bait and untagged Rab8a as prey. Rab8a species were either non-phosphorylated Rab8a-GTP(Q67L), pRab8a-GTP(Q67L), or pRab8a(GDP). Protein concentrations were 10µM for bait and prey, inputs are 2 µg; n≥3, Coomassie stain for visualization. Dotted lines emphasize that only pRab8a(GTP) binds to RILPL2. B , HEK293 cells were transiently transfected with constructs expressing the indicated components. 24 h post-transfection, cells were treated with ± 100 nM MLi-2 for 90 min and then lysed. Upper-panel labelled IP:GFP, RILPL2-GFP was immunoprecipitated using GFP binder sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx Western Blot imaging system analysis with the indicated antibodies at 0.5-1 µg/mL concentration. Lower-panel labelled Input-10 µg whole cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E.coli expression.

Techniques: In Vitro, Purification, Staining, Transfection, Construct, Expressing, Immunoprecipitation, Western Blot, Imaging, Concentration Assay