First 3D-Structural Data of Full-length Rod-Outer-Segment Guanylyl Cyclase 1 in Bovine Retina by Cross-linking/Mass Spectrometry
Christian H. Ihling,
Posted 25 Sep 2020
bioRxiv DOI: 10.1101/2020.09.25.312835
Posted 25 Sep 2020
The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 in bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data reveal that the α-helical domain connecting the kinase homology and catalytic domains can acquire different conformations. Also, the XL-MS data of native ROS-GC1 in bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment. ### Competing Interest Statement The authors have declared no competing interest. * αHD : α-helical domain ACN : Acetonitrile ATP : Adenosine triphosphate CD : Catalytic domain CCD : Core catalytic domain cGMP : Cyclic guanosine monophosphate CORD : Cone-rod dystrophies CTE : C -terminal extension DD : Dimerization domain DDM : n -Dodecyl-β-D-maltopyranoside DMEM : Dulbecco’s Modified Eagle’s Medium DMSO : Dimethyl sulfoxide DSBU : Disuccinimidyl dibutyric urea DTT : Dithiothreitol EDTA : 2,2’,2”,2ū-(Ethane-1,2-diyldinitrilo)tetraacetic acid EGTA : Ethylene glycol-bis(2-aminoethylether)- N,N,N’,N -tetraacetic acid ESI : Electrospray ionization ExtD : Extracellular domain FA : Formic acid FASP : Filter-aided sample preparation GC : Guanylyl cyclase GCAP : Guanylyl cyclase-activating protein GTP : Guanosine triphosphate GMP : Guanosine monophosphate HCD : Higher energy collision-induced dissociation HEK : Human embryonic kidney HEPES : 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HLT : (His)8 tag, lipoyl domain, and tobacco etch virus cleavage site IAA : Iodoacetamide IBMX : 3-isobutyl-1-methylxanthine IcD : Intracellular domain IPTG : Isopropyl β-D-1-thiogalactopyranoside JMD : Juxtamembrane domain KHD : Kinase homology domain LC : Liquid chromatography LCA : Leber’s congenital amourosis LS : Leader sequence MRM : Multiple reaction monitoring MS : Mass spectrometry MS/MS : Tandem mass spectrometry NPR : Natriuretic peptide receptor NTA : Nitrilotriacetic acid ROS : Rod-outer-segment RP : Reversed phase SCAD : Surfactant and chaotropic agent-assisted sequential extraction/on-pellet digestion SDS-PAGE : Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEC : Size exclusion chromatography SHD : Signal helix domain SRM : Selected reaction monitoring STaR : Heat-stable enterotoxin receptor TFA : Trifluoroacetic acid Th : Thomson TMD : Transmembrane domain Tris : Tris(hydroxymethyl)aminomethane XL-MS : Cross-linking/mass spectrometry
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