Abstract
The protein Rgd1 is involved in the regulation of cytoskeleton formation and in signalling pathways that control cell polarity and growth in Saccharomyces cerevisiae. Rgd1p is composed of a F-BAR domain required for membrane binding and a RhoGAP domain responsible for activating Rho3p and Rho4p, two GTPases respectively involved in bud growth and cytokinesis. Rgd1p is recruited to the membrane through interactions with phosphoinositide lipids, which bind the two isolated domains and stimulate the RhoGAP activity on Rho4p. As previously shown by crystallography, the membrane-binding F-BAR domain contains a conserved inositol phosphate binding site, which explains the preferential binding of phosphoinositides. In contrast, RhoGAP domains are not expected to bind lipids. In order to unravel this puzzling feature, we solved the three-dimensional structure of the isolated protein and found a cryptic phosphoinositide binding site involving non conserved residues (Martinez et al. 2017). The assignment of the resonances and secondary structure of Rgd1-RhoGAP (aa 450–666) is presented here.
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Abbreviations
- BAR:
-
Bin-Amphiphysin-Rvs (BAR)
- DLC1:
-
Deleted in liver cancer 1
- F-BAR:
-
F-BAR FCH, and BAR; (FCH = fes, CIP4 homology)
- G protein:
-
Guanine nucleotide-binding proteins
- HSQC:
-
Heteronuclear single quantum coherence transfer
- Ni–NTA:
-
Nickel–nitrilotriacetic acid
- PtdIns(3,4,5)P3 :
-
Phosphatidylinositol-3,4,5-trisphosphate
- PtdIns(4,5)P2 :
-
Phosphatidylinositol-4,5-bisphosphate
- PtdIns(4)P:
-
Phosphatidylinositol-4-phosphate
- Rgd1-RhoGAP:
-
RhoGAP domain from related GAP domain 1 protein
- Rho:
-
Ras homology
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Acknowledgements
We thank the TGIR-RMN-THC Fr3050 CNRS and the structural biology platform at the Institut Européen de Chimie et Biologie (UMS 3033) for access to NMR spectrometers and technical assistance. The authors are grateful to Annie Clavères for expert technical assistance. D. M. was supported by a French PhD fellowship afforded to the University of Bordeaux by the Ministère de la Recherche (MNERT).
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Martinez, D., Prouzet-Mauléon, V., Hugues, M. et al. Assignment of 1H, 13C and 15N resonances and secondary structure of the Rgd1-RhoGAP domain. Biomol NMR Assign 12, 129–132 (2018). https://doi.org/10.1007/s12104-017-9794-z
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DOI: https://doi.org/10.1007/s12104-017-9794-z