Mapping the isoprenoid binding pocket of PDEdelta by a semisynthetic, photoactivatable N-Ras lipoprotein.
Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The delta subunit of phosphodiesterase (PDEdelta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEdelta can be mapped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of beta-sheet 9 of PDEdelta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEdelta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEdelta.
Department of Structural Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn Strasse 11, Dortmund, Germany.
Michael
M
Alexander
Marc
M
Gerauer
Markos
M
Pechlivanis
Boriana
B
Popkirova
Radovan
R
Dvorsky
Luc
L
Brunsveld
Herbert
H
Waldmann
Jürgen
J
Kuhlmann
D000595:Amino Acid Sequence; D001665:Binding Sites; D019295:Computational Biology; D003432:Cross-Linking Reagents / Q000378:metabolism; D006801:Humans; D008027:Light; D008074:Lipoproteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013729:Terpenes / Q000378:metabolism*; D018631:ras Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008074:Lipoproteins; D013729:Terpenes; D010727:Phosphoric Diester Hydrolases; D018631:ras Proteins