Structural and functional characterization of the interaction between 2',3'-dialdehyde guanine nucleotide analogues and the stimulatory G protein alpha-subunit.
We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.
Hohenegger
M
M
Institute of Pharmacology, University of Vienna, Austria.
Nanoff
C
C
Ahorn
H
H
Freissmuth
M
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002540:Cerebral Cortex / Q000737:chemistry; D004252:DNA Mutational Analysis; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D006150:Guanine Nucleotides; D008024:Ligands; D010446:Peptide Fragments; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037756:2-(guanylformylmethoxy)-3-(triphospho)propanal; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases