G protein-bound conformation of mastoparan-X: heteronuclear multidimensional transferred nuclear overhauser effect analysis of peptide uniformly enriched with 13C and 15N.
Mastoparans, a family of tetradecapeptides from wasp venom, have been used as convenient low molecular weight models of receptors coupled to GTP-binding regulatory proteins (G proteins) for the understanding of the interaction between G proteins and receptors. Sukumar and Higashijima have analyzed the conformation of mastoparan-X (MP-X) bound to the G protein alpha-subunit using proton two-dimensional transferred nuclear Overhauser effect (TRNOE) spectroscopy [Sukumar, M., and Higashijima, T. (1992) J. Biol. Chem., 267, 21421-21424]. The resultant structure, however, was not well-defined due to severe overlap of peptide proton resonances. To determine the G protein-bound conformation of MP-X in detail, we have analyzed this interaction by heteronuclear multidimensional TRNOE experiments of MP-X uniformly enriched with 15N and/or 13C. By solving the overlap problem, we were able to determine the precise conformation of MP-X bound to Gi1alpha: the peptide adopts an amphiphilic alpha-helix from Trp3 to C-terminal Leu14, and the atomic root-mean-square deviation (rmsd) values in this portion about the averaged coordinates were 0.27 +/- 0.07 A for the backbone atoms (N, Calpha, C') and 0.84 +/- 0.16 A for all heavy atoms. These values are much smaller than the corresponding rmsd values of the structures obtained from the proton 2D TRNOE spectrum alone: 1.70 +/- 0.41 A for the backbone atoms (N, Calpha, C') and 2.84 +/- 0.51 A for all heavy atoms. Our results indicate that the heteronuclear multidimensional TRNOE experiments of peptides uniformly enriched with stable isotopes are a very powerful tool for analyzing the conformation of short peptides bound to large proteins. We will also discuss the structure-activity relationships of mastoparans in activating G proteins on the basis of the precise structure of MP-X bound to Gi1alpha.
Kusunoki
H
H
Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Machida-shi, Tokyo 194, Japan.
Wakamatsu
K
K
Sato
K
K
Miyazawa
T
T
Kohno
T
T
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D055672:Static Electricity
D002247:Carbon Isotopes; D036341:Intercellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes; D010455:Peptides; C071123:mastoparan X; D019204:GTP-Binding Proteins