Structural and functional characterization of the interaction between 2',3'-dialdehyde guanine nucleotide analogues and the stimulatory G protein alpha-subunit.

摘要: Abstract We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing ability periodate-oxidized 2',3'-dialdehyde analogues GTP (oGTP) and gamma S (oGTP S) to bind recombinant alpha-subunit stimulatory protein, rGs alpha-s. oGTP alpha-s in a quasi-irreversible manner via formation Schiff's base, can be reduced with borhydrid resulting covalent incorporation [alpha-32P]oGTP [35S]oGTP into When bound alpha-s, is hydrolyzed traps inactive conformation, while persistently activates alpha. Thus, act as antagonist agonist, respectively, represent pair suitable functional structural tools. Cleavage covalently labeled cyanogen bromide generates several fragments. Labeled fragments were assigned G1 G4 region using sequence-specific antisera. An additional, fragment was identified amino-terminal sequencing corresponded helix alpha A recently determined crystal structure transducin (Noel, J. P., Hamm, H. E., Sigler, P. B. (1993) Nature 366, 654-663). In oGDP-liganded occurs predominantly G1-fragment, labels additional similar extent indicating tight packing around active conformation. Furthermore, contains single acid cleavable bond (Asp317-Pro318), such that formic releases carboxyl-terminal from [alpha-32P]oGTP- S-liganded This lysine residue (Lys324) only S. Lys324 unique Gs lies within its effector region. Hence, during switch state, this undergoes major conformational change moves it closer pocket.