Conformation of a Cdc42/Rac Interactive Binding Peptide in Complex with Cdc42 and Analysis of the Binding Interface
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- 作者：Willem K. Stevens ; Wim Vranken ; Nathalie Goudreau ; Hui Xiang ; Ping Xu ; and Feng Ni
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：May 11, 1999
- 年：1999
- 卷：38
- 期：19
- 页码：5968 - 5975
- 全文大小：107K
- 年卷期：v.38,no.19(May 11, 1999)
- ISSN：1520-4995
文摘
Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a commonsequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with aconsensus of I-S-x-P-(x)2-4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071-29074], has been shown to be essential for the functional interactions between these effector proteins andCdc42. We have characterized the interactions of a 22-residue CRIB peptide derived from human PAK2[PAK2(71-92)] with Cdc42 using proton and heteronuclear NMR spectroscopy. This CRIB peptide bindsto GTP-
S-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 
M, as determined byfluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92)with GTP-S-loaded Cdc42 causes resonance perturbations in the 1H-15N HSQC spectrum of Cdc42that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo,W., et al. (1998) Biochemistry 37, 14030-14037]. Proton NMR studies of PAK2(71-92) demonstratestructuring of PAK2(71-92) in the presence of GTP-S-loaded Cdc42, through the observation of manynonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs showthat the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the sidechains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. TheCRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and itseffector proteins.
S-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 M, as determined byfluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92)with GTP-S-loaded Cdc42 causes resonance perturbations in the 1H-15N HSQC spectrum of Cdc42that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo,W., et al. (1998) Biochemistry 37, 14030-14037]. Proton NMR studies of PAK2(71-92) demonstratestructuring of PAK2(71-92) in the presence of GTP-S-loaded Cdc42, through the observation of manynonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs showthat the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the sidechains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. TheCRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and itseffector proteins.