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Collection : Elife (E-Journal - Via Crossref)

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Figure 3. Notable contacts to the PRPP ligand in chain A. PRPP is depicted as sticks and colored by element with purple carbons. ; Nucleotides are depicted as sticks and colored by element with blue carbons. Individual nucleotides and metals are labeled. Dashed black lines indicate hydrogen bonds. A dashed green line shows the lone pair-π interaction between A103 and G105. Solid black lines indicate coordination to a metal ion. Relative to Figure 1, the structure is rotated 180° about the y axis. Panel A is additionally rotated nearly 90° about the x axis. Panel D is rotated approximately 45° about the x axis in the opposite direction. (A) Contacts among the 5-phosphate of PRPP, residues G48, C49, C77, and metal ions M1 and M3. (B) Hydrogen bonds between the ribose of PRPP and residues C77, G96, and G104. (C) Coordination of metal M2 by PRPP and residue G6. (D) Recognition of the pyrophosphate group of PRPP by residues A5, G6, A101, and G105.

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Figure 4. Rasip1 binds to HEG1 upstream of the KRIT1-binding site. ; (A) Schematic representation of different HEG1 cytoplasmic tail peptides used to map the binding region for Rasip1. (B) HEK293T cells were transfected with GFP-tagged full-length Rasip1. Western blot analysis shows that HEG1 wild-type (WT), 1334X, C54, and 1318-1339 bound to GFP-Rasip1. In contrast, HEG1 1328X, C49, and ∆1327-1335 failed to bind to GFP-Rasip1. Endogenous KRIT1 binding was only observed for HEG1 WT, C54, C49, and ∆1327-1335 which al contain the C-terminal YF motif. Affinity Matrix was visualized by Ponceau staining. (C) Top section: Bar diagram shows binding of GFP-Rasip1 to HEG1 cytoplasmic tail peptides relative to wild-type HEG1. Mean values ± SEM are shown from at least 3 independent experiments. Bottom section: HEG1 1327-1335 (TDVYYSPTS) is necessary for Rasip1 binding. (D) HUVECs, transfected with mito-mCherry-HEG1 or mito-mCherry-HEG1(∆1327-1335), were analyzed by Spinning Disk Confocal Miroscopy (SDCM) for endogenous Rasip1 localization. A fraction of Rasip1 was targeted to mito-mCherry-HEG1 positive structures but not to mito-mCherry-HEG1(∆1327-1335). Scale bars, 10 µm. (E) HEK293T cells were transfected with GFP-tagged full-length Rasip1, FLAG-tagged murine HEG1 full-length, ∆1283-1291 (corresponding to aa 1327-–1335 in human HEG1), empty vector, or both. Immunoprecipitation was done by using anti-FLAG G1 resin and bound proteins were separated by SDS-PAGE. Western blot analysis shows that GFP-tagged Rasip1 was co-immunoprecipitated with full-length mHEG1 but not mHEG1(∆1283-–1291).

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