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Biomolecular NMR Assignments

, Volume 12, Issue 1, pp 129–132 | Cite as

Assignment of 1H, 13C and 15N resonances and secondary structure of the Rgd1-RhoGAP domain

  • Denis Martinez
  • Valérie Prouzet-Mauléon
  • Michel Hugues
  • François Doignon
  • Benoît Odaert
Article

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.

Keywords

RhoGAP Rgd1 Assignment NMR Phosphoinositide 

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

Notes

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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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Chimie et Biologie des Membranes et des Nano-objetsUniversité de Bordeaux, CNRS UMR 5248Pessac CedexFrance
  2. 2.Laboratoire d’Hématopoïèse Leucémique et Cibles ThérapeutiquesUniversité de Bordeaux, INSERM 1035Bordeaux CedexFrance
  3. 3.Laboratoire de Biogenèse MembranaireUniversité de Bordeaux, CNRS UMR 5200Villenave d’Ornon CedexFrance

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