Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin.
The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.
Aris
L
L
Institute for Neuroscience, Northwestern University, Chicago, Illinois 60611, USA.
Gilchrist
A
A
Rens-Domiano
S
S
Meyer
C
C
Schatz
P J
PJ
Dratz
E A
EA
Hamm
H E
HE
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003545:Cysteine; D005998:Glycine; D008239:Lysine; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D021122:Protein Subunits; C013110:metarhodopsins; D012243:Rhodopsin; D015293:Transducin; D008239:Lysine; D003545:Cysteine; D005998:Glycine