PMC:6174286 / 13874-15890
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6174286","sourcedb":"PMC","sourceid":"6174286","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6174286","text":"Figure 3 The Human ARL13B-ARL3 Complex Is Predicted to Involve an Interaction between Evolutionarily Conserved Glutamate and Arginine Residues\n(A) Partial amino acid sequence alignments of the ciliary GEF, ARL13B, and ARL3. Highlighted in red are the evolutionarily conserved glutamate residue located in the switch II domain of ARL13B (E86 [Glu86] in C. reinhardtii ARL13B [CrARL13B]) and the arginine residue in the loop region between the α4 and β6 domains of ARL3 (R148 [Arg148] in CrARL3).\n(B) Superimposition of the crystal structures of ARL3 (gray) in complex with its known interactors: the effectors UNC119A (salmon; PDB: 4GOJ15) and BARTL1 (yellow; PDB: 4ZI221), the GAP RP2 (orange; PDB: 3BH617), and GEF ARL13B (blue; PDB: 5DI319). On the right side is a zoomed-in view of the salt bridge between Glu86 and Arg148 at the CrARL13B-CrARL3 complex interface.\n(C) Assay of GEF activity for murine WT ARL3 (ARL3WT) and p.Arg149His ARL3 (ARL3R149H). Fluorescence polarization was measured for 1 μM mantGDP-loaded ARL3, to which 400 μM GppNHp and 5 μM H. sapiens ARL13B (HsARL13B) were added. Nucleotide exchange was shown by only ARL3WT.\n(D) Assay of GEF activity with fluorescence polarization measurements of 0.5 μM mantGDP-loaded CrARL3WT and CrARL3R148H, to which 10 μM GppNHp (GTP analog) and 5 μM CrARL13B⋅GppNHp were added at the indicated time points. Only CrARL3WT showed nucleotide exchange, as indicated by the drop in fluorescence polarization.\n(E) Assay of GEF activity with fluorescence polarization measurements of 0.5 μM mantGDP-loaded CrARL3WT and 5 μM CrARL13B⋅GppNHpWT or CrArl13bE86R, to which 10 μM GppNHp (GTP analog) was added at the indicated time points. Only CrArl13bWT showed nucleotide exchange, as indicated by the drop in fluorescence polarization.\n(F) 30 μg of full-length UNC119A-GST was used to pull down 60 μg of murine ARL3WT and ARL3R149H that were loaded with the GTP analog GppNHp. Proteins were detected on immunoblots with anti-GST (red) and anti-His (green) antibodies.","divisions":[{"label":"label","span":{"begin":0,"end":8}},{"label":"p","span":{"begin":9,"end":142}},{"label":"p","span":{"begin":143,"end":494}},{"label":"p","span":{"begin":495,"end":867}},{"label":"p","span":{"begin":868,"end":1143}},{"label":"p","span":{"begin":1144,"end":1462}},{"label":"p","span":{"begin":1463,"end":1784}}],"tracks":[{"project":"2_test","denotations":[{"id":"30269812-22960633-2052037","span":{"begin":635,"end":637},"obj":"22960633"},{"id":"30269812-26455799-2052038","span":{"begin":667,"end":670},"obj":"26455799"},{"id":"30269812-18376416-2052039","span":{"begin":702,"end":705},"obj":"18376416"},{"id":"30269812-26551564-2052040","span":{"begin":738,"end":741},"obj":"26551564"}],"attributes":[{"subj":"30269812-22960633-2052037","pred":"source","obj":"2_test"},{"subj":"30269812-26455799-2052038","pred":"source","obj":"2_test"},{"subj":"30269812-18376416-2052039","pred":"source","obj":"2_test"},{"subj":"30269812-26551564-2052040","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#c4ec93","default":true}]}]}}