Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p–F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p–RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1′ and B helices. The addition of helix A1′, unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p–RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.
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Cover Image
The 26S proteasome. Image kindly provided by Professor Joel Kowit from his work Degradation, a depiction in stained glass of the 26S proteasome (see http://joelkowit.com). In this issue of the Biochemical Journal, VerPlank and Goldberg describe how phosphorylation of the proteasome regulates protein breakdown by reviewing the ability of several kinases to alter proteasome function; see pages 3355–3371 for further details.
Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain
Denis Martinez, Béatrice Langlois d'Estaintot, Thierry Granier, James Tolchard, Cécile Courrèges, Valérie Prouzet-Mauléon, Michel Hugues, Bernard Gallois, François Doignon, Benoît Odaert; Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain. Biochem J 1 October 2017; 474 (19): 3307–3319. doi: https://doi.org/10.1042/BCJ20170331
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