STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE INTERACTION BETWEEN2',3'-DIALDEHYDE GUANINE-NUCLEOTIDE ANALOGS AND THE STIMULATORY G-PROTEIN ALPHA-SUBUNIT

We have searched for irreversible ligands which target the guanine nuc leotide binding pocket of G protein alpha-subunits by testing the abil ity of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogue s of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombin ant cy-subunit of the stimulatory G protein, rG(s alpha-s). oGTP and o GTP gamma S bind to rG(s alpha-s) in a quasi-irreversible manner via f ormation of a Schiff's base, which can be reduced with borhydrid resul ting in covalent incorporation of [alpha-P-32]oGTP and [S-35]oGTP gamm a S into rG(s alpha-s). When bound to rG(s alpha), oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rG(s alpha). Thus, oGTP and oGTP gamma S act a s irreversible G protein antagonist and agonist, respectively, and rep resent a pair of nucleotide analogues suitable as functional and struc tural tools, Cleavage of covalently Iabeled rG(s alpha-s) with cyanoge n bromide generates several labeled fragments. Labeled fragments were assigned to the G(1) and G(4) region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragm ent was identified by amino-terminal sequencing and corresponded to th e helix alpha A in the recently determined crystal structure of the tr ansducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1 993) Nature 366, 654-663), In the oGDP-liganded conformation, incorpor ation occurs predominantly into the G(1)-fragment, while [S-35]oGTP ga mma S labels the additional fragments to a similar extent indicating t ight packing around the guanine nucleotide binding pocket in the activ e conformation, Furthermore, rG(s alpha-s) contains a single acid clea vable bond (Asp(317)-pro(318)), such that formic acid releases a carbo xyl-terminal fragment from [alpha(32)PGTP- and [S-35]oGTP gamma S-liga nded rG(s alpha-s). This fragment contains a single lysine residue (Ly s(324)) which is only labeled by [S-35]oGTP gamma S. Lys(324) is uniqu e to G(s alpha) and lies within its effector binding region, Hence, du ring the switch from the inactive to the active state, this region und ergoes a major conformational change that moves it closer to the nucle otide binding pocket.