Regional Polysterism in the GTP-Bound Form of the Human c-Ha-Ras Protein
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- 作者：Yutaka Ito ; Kazuhiko Yamasaki ; Junji Iwahara ; Tohru Terada ; Akihide Kamiya ; Mikako Shirouzu ; Yutaka Muto ; Gota Kawai ; Shigeyuki Yokoyama ; Ernest D. Laue ; Markus Wä ; lchli ; Takehiko Shibata ; Susumu
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：July 29, 1997
- 年：1997
- 卷：36
- 期：30
- 页码：9109 - 9119
- 全文大小：398K
- 年卷期：v.36,no.30(July 29, 1997)
- ISSN：1520-4995
文摘
The backbone 1H, 13C, and 15Nresonances of the c-Ha-Ras protein [a truncated versionconsisting of residues 1-171, Ras(1-171)] bound with GMPPNP(a slowly hydrolyzable analogue ofGTP) were assigned and compared with those of the GDP-boundRas(1-171). The backbone amideresonances of amino acid residues 10-13, 21, 31-39, 57-64, and 71of Ras(1-171)·GMPPNP, but notthose of Ras(1-171)·GDP, were extremely broadened,whereas other residues of Ras(1-171)·GMPPNPexhibited amide resonances nearly as sharp as those ofRas(1-171)·GDP. The residues exhibitingtheextreme broadening, except for residues 21 and 71, are localized inthree functional loop regions [loopsL1, L2 (switch I), and L4 (switch II)], which are involved inhydrolysis of GTP and interactions withother proteins. From the temperature and magnetic field strengthdependencies of the backbone amideresonance intensities, the extreme broadening was ascribed to theexchange at an intermediate rate on theNMR time scale. It was shown that the Ras(1-171) proteinbound with GTP or GTP
S (another slowlyhydrolyzable analogue of GTP) exhibits the same type of broadening.Therefore, it is a characteristicfeature of the GTP-bound form of Ras that the L1, L2, and L4 loopregions, but not other regions, are ina rather slow interconversion between two or more stable conformers.This phenomenon, termed a "regionalpolysterism", of these loop regions may be related with theirmultifunctionality: the GTP-dependentinteractions with several downstream target groups such as the Raf andRalGDS families and also withthe GTPase activating protein (GAP) family. In fact, the bindingof Ras(1-171)·GMPPNP with theRas-binding domain (residues 51-131) of c-Raf-1 was shown toeliminate the regional polysterism nearlycompletely. It was indicated, therefore, that eachtarget/regulator selects its appropriate conformer amongthose presented by the "polysteric" binding interface of Ras.As the downstream target groups exhibit noapparent sequence homology to each other, it is possible that onetarget group prefers a conformer differentfrom that preferred by another group. The involvement of loop L1in the regional polysterism mightsuggest that the negative regulators, GAPs, bind to the polystericbinding interface (loops L2 and L4) ofRas and cooperatively select a conformer suitable for transition of theGTPase catalytic center, involvingloops L1 and L4, into the highly active state.
S (another slowlyhydrolyzable analogue of GTP) exhibits the same type of broadening.Therefore, it is a characteristicfeature of the GTP-bound form of Ras that the L1, L2, and L4 loopregions, but not other regions, are ina rather slow interconversion between two or more stable conformers.This phenomenon, termed a "regionalpolysterism", of these loop regions may be related with theirmultifunctionality: the GTP-dependentinteractions with several downstream target groups such as the Raf andRalGDS families and also withthe GTPase activating protein (GAP) family. In fact, the bindingof Ras(1-171)·GMPPNP with theRas-binding domain (residues 51-131) of c-Raf-1 was shown toeliminate the regional polysterism nearlycompletely. It was indicated, therefore, that eachtarget/regulator selects its appropriate conformer amongthose presented by the "polysteric" binding interface of Ras.As the downstream target groups exhibit noapparent sequence homology to each other, it is possible that onetarget group prefers a conformer differentfrom that preferred by another group. The involvement of loop L1in the regional polysterism mightsuggest that the negative regulators, GAPs, bind to the polystericbinding interface (loops L2 and L4) ofRas and cooperatively select a conformer suitable for transition of theGTPase catalytic center, involvingloops L1 and L4, into the highly active state.