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. 2014 Jul 29;426(15):2755-68.
doi: 10.1016/j.jmb.2014.05.011. Epub 2014 May 29.

Molecular conformation of the full-length tumor suppressor NF2/Merlin--a small-angle neutron scattering study

Jahan Ali Khajeh  1 , Jeong Ho Ju  1 , Moussoubaou Atchiba  1 , Marc Allaire  2 , Christopher Stanley  3 , William T Heller  3 , David J E Callaway  1 , Zimei Bu  4
Affiliations

Molecular conformation of the full-length tumor suppressor NF2/Merlin--a small-angle neutron scattering study

Jahan Ali Khajeh et al. J Mol Biol. .

Abstract

The tumor suppressor protein Merlin inhibits cell proliferation upon establishing cell-cell contacts. Because Merlin has high level of sequence similarity to the Ezrin-Radixin-Moesin family of proteins, the structural model of Ezrin-Radixin-Moesin protein autoinhibition and cycling between closed/resting and open/active conformational states is often employed to explain Merlin function. However, recent biochemical studies suggest alternative molecular models of Merlin function. Here, we have determined the low-resolution molecular structure and binding activity of Merlin and a Merlin(S518D) mutant that mimics the inactivating phosphorylation at S518 using small-angle neutron scattering and binding experiments. Small-angle neutron scattering shows that, in solution, both Merlin and Merlin(S518D) adopt a closed conformation, but binding experiments indicate that a significant fraction of either Merlin or Merlin(S518D) is capable of binding to the target protein NHERF1. Upon binding to the phosphatidylinositol 4,5-bisphosphate lipid, the wild-type Merlin adopts a more open conformation than in solution, but Merlin(S518D) remains in a closed conformation. This study supports a rheostat model of Merlin in NHERF1 binding and contributes to resolving a controversy about the molecular conformation and binding activity of Merlin.

Keywords: Ezrin; Merlin; neurofibromatosis type 2; phosphatidylinositol 4,5-bisphosphate; small-angle neutron scattering.

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Figures

Figure 1
Figure 1. Purified Merlin and Merlin(S518D) are monomeric
(A) SDS-PAGE of purified Merlin(wt), Merlin(S518D), and Merlin(S518E). (B) Gel filtration of Merlin(wt), Merlin(S518D), and Merlin(S518E). The gel filtration was performed at room temperature. (C) Dynamic light scattering of Merlin(wt) and Merlin(S518D). Left panel: auto-correlation function. Right panel: size distribution. Molecular mass measurements by static light scattering indicate that Merlin and Merlin(S518D) are monomers. The hydrodynamic radius Rh=48.4±0.5 Å for Merlin(wt), and Rh=47.9±0.1 Å for Merlin(S518D). The light scattering experiments were performed at 25 °C.
Figure 2
Figure 2. Comparing binding affinities of NHERF1 for ezFERM, mFERM, full-length Merlin(wt), and Merlin(S518D), respectively
(A) SPR experiments were performed with NHERF1 immobilized on a CM5 sensor chip. Merlin constructs and ezFERM at different concentrations were flown on the NHERF1 immobilized sensor chip. (B) ITC experiments to determine the binding of NHERF1 to ezFERM, mFERM, Merlin(wt), and Melrin(S518D). The binding results are summarized in Table 2.
Figure 2
Figure 2. Comparing binding affinities of NHERF1 for ezFERM, mFERM, full-length Merlin(wt), and Merlin(S518D), respectively
(A) SPR experiments were performed with NHERF1 immobilized on a CM5 sensor chip. Merlin constructs and ezFERM at different concentrations were flown on the NHERF1 immobilized sensor chip. (B) ITC experiments to determine the binding of NHERF1 to ezFERM, mFERM, Merlin(wt), and Melrin(S518D). The binding results are summarized in Table 2.
Figure 3
Figure 3. Comparing the SANS data of dMerlin(wt), dMerlin(S518D) and dEzrin(wt0 in solution
(A) SANS data of dMerlin, dMerlin(S518D), and dEzrin in H2O buffer solution. (B) Guinier plots of the data shown in (A). (C) P(r) functions of dMerlin(wt), dMerlin(S518D), and dEzrin. (D) 3-D shapes of dMerlin(wt), dMerlin(S518D), and dEzrin reconstructed from SANS. The crystal structure Moesin (PDB code: 2I1K)[22] is docked in the 3-D shapes as a comparison. The SANS experiments were performed on 2.2 mg/ml dMerlin and 1.7 mg/ml dMerlin(S518D), and on 1.8 mg/ml dEzrin(wt) in solution [32].
Figure 4
Figure 4. ITC experiments to determine the binding of PIP2 to ezFERM, mFERM, Merlin(wt), and Merlin(S518D)
PIP2 solution of 900 μM was titrated into the protein solutions of 30 μM. The binding data are summarized in Table 3.
Figure 5
Figure 5. PIP2-binding induces the dMerlin(wt) to adopt a more open conformation than in solution, but dMerlin(S518D) is retained in a closed conformation
(A) SANS data of the dMerlin(wt) at 1.3 mg/ml and dMerlin(S518D) at 1.1 mg/ml, and the PIP2-bound dMerlin(wt) at 0.8 mg/ml and dMerlin(S518D) at 0.7 mg/ml in 20% D2O at the contrast-matching point of PIP2. (B) Guinier plots of SANS data shown in (A). (C) Comparing P(r) functions of dMerlin(wt) and dMerlin(S518D) in solution and in PIP2. (D) Comparing the Rg and Dmax distribution of dMerlin(wt) and dMerlin(S518D) in solution and in PIP2.
Figure 6
Figure 6
Comparing the changes in 3-D shapes of (A) dMerlin(wt) (A) and (B) dMerlin(S518D) in PIP2 (lower panel). The 3-D images are reconstructed from the SANS data using DAMMIN [41]. Note that the fit to the envelope in (A) is a simplification, as the C-terminal tail domain is not necessarily folded in absence of interaction with FERM domain.
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