A novel bipartite phospholipid-binding module in the neurofibromatosis type 1 protein

Abstract

Neurofibromatosis type 1 (NF1) is a common tumour predisposition syndrome associated with numerous clinical complications. Mutations in the tumour suppressor gene NF1 are responsible for disease pathogenesis. This gene encodes the 320 kDa protein neurofibromin, the only clearly defined function of which is to act as a Ras-specific GTPase-activating protein (RasGAP). Here we report the structural discovery of a novel module in neurofibromin, composed of a Sec14p homologous segment and a previously undetected pleckstrin homology (PH)-like domain of potentially novel function. We show phospholipid binding by this bipartite module and identify residues that are involved in this activity; we also show that the PH-like domain is not sufficient for lipid binding. The unique architecture of the domain interface points to a model of how the PH-like domain may regulate binding of a ligand by the Sec14 module.

Figure 1

Structure and phospholipid binding of the NF1-Sec-PH module. (A) Ribbon representation of the NF1-Sec-PH module of the human neurofibromin, clearly showing the two domain architecture. (B) Close-up view of the domain interface with selected side chains of basic residues included in ball and stick. The β-protrusion of NF1-PH seems to stabilize a closed conformation by contact with the lid–helix of NF1-Sec. (C) Overlay assays with NF1-Sec-PH and membranes spotted with 100 pmol phospholipids (left panel), indicating binding selecting for phosphorylated phosphatidylinositol (PI). Overlay assays (PIP-array™) spotted with increasing amounts of phosphorylated PI (middle panel), suggesting preference for monophosphorylated species. Overlay assays spotted with 100 pmol of various lipid compounds (Sphingo-Strips™, right panel) show that sulphatide is the preferred binder in this assay, with minor signal derived from lysophosphatidic acid (LPA).

Figure 2

Structure-based sequence alignment of the NF1-Sec-PH region showing similarities with other neurofibromin orthologues as well as with disease-related Sec14p-like proteins (NF1-Sec only). Sequence identity is highlighted in red and similarity in yellow. Structure-based mutations are indicated with black circles; disease-related mutations are indicated with green-filled circles (see Fig 3A). The resulting mutation is specified in bold writing. Sequence source codes (Uniprot): Hs_NF1-I, Homo sapiens neurofibromin (P21359-2); Dm_NF1, D. melanogaster neurofibromin (O01397); Nc_NF1, Neurospora crassa NF1 homologue (Q8WZX6); Dd_NF1, Dictyostelium discoideum NF1 homologue (Q8MNG1); Sc_IRA1, Saccharomyces cerevisiae IRA1 (P18963); Sc_IRA2: S. cerevisiae IRA2 (P19158); Sc_Sec14p: S. cerevisiae Sec14p (P24280); Hs_attp: Homo sapiens α-tocopherol-transfer protein (P49638); Hs_TAP/SPF: H. sapiens supernatant protein factor (SPF) (O76054); Hs_ATCAY: H. sapiens caytaxin (Q86WG3). The tandem duplication detected in Noonan's syndrome patients and its insertion position is indicated; the duplicated span corresponds to the linker region between the pleckstrin homology (PH) and Sec domains.

Figure 3

Biochemical analysis of phospholipid binding to NF1-Sec-PH by site-directed mutagenesis along with phospholipid-overlay assays. (A) Top view of the NF1-Sec-PH model with positions of mutated residues indicated with dark grey spheres. Positions of missense mutations identified in NF1 patients are indicated by green spheres and deletions by a green X. The region of the tandem duplication in the domain linker is also indicated. (B) Overlay assays (PIP-arrays™ and Sphingo-Strips™) performed with various mutants and the GST-fused NF1-PH (bottom right panel; see text).

Figure 4

Proposed functional mechanism of the NF1-SEC-PH module. (A) Ribbon diagram of NF1-Sec-PH showing superimposed open (derived from Sec14p; Sha et al, 1998) and closed conformation (this structure). It is clearly visible that the open conformation would clash with the β-protrusion, making us postulate conformational rearrangements in this region to allow movement of the lid–helix region. (B) Hypothetical mechanism of how conformational changes in the NF1-PH domain may regulate access to the NF1-Sec lipid-binding cage. The putative regulatory ligand A could be a phospholipid, but the possibility of other types of molecule including proteins has to be considered (see text). On interaction with ligand A, conformational changes involving the β-protrusion of the NF1-PH domain and the lid–helix of the NF1-Sec module may control binding of ligand B, which may be a membrane lipid binding inside the NF1-Sec. (C) Structurally validated domain scheme of neurofibromin with domain boundaries indicated.