Electrostatic interaction between oxysterol-binding protein and VAMP-associated protein A revealed by NMR and mutagenesis studies - PubMed (original) (raw)

Electrostatic interaction between oxysterol-binding protein and VAMP-associated protein A revealed by NMR and mutagenesis studies

Kyoko Furuita et al. J Biol Chem. 2010.

Abstract

Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBP(F)) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBP(F), and the complex. Our results show that OSBP(F) is disordered in the free state, and VAP-A(MSP) and OSBP(F) form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain.

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Figures

FIGURE 1.

FIGURE 1.

A, domain structures of human VAP-A and human OSBP are shown. MSP, major sperm protein; CC, coiled-coil; TM, transmembrane; PH, pleckstrin homology; LB, lipid binding. B, alignment of FFAT motifs of human lipid binding proteins and rat ORP1 is shown.

FIGURE 2.

FIGURE 2.

Solution structure of the complex between VAP-A (6–125) and OSBP (358–366). A, shown is a superimposed representation of 20 lowest energy structures. B, shown is a ribbon representation of VAP-AMSP. OSBPF is shown as a stick model. C, shown are the electrostatic surfaces of VAP-AMSP (left) and OSBPF (right) contoured from −3 kT (red) to +3 kT (blue). The electrostatic potential was calculated using an amber force field.

FIGURE 3.

FIGURE 3.

Details of the interaction. Residue numbers of OSBPF are written in italics. OSBPF is shown as a stick model. The surface of VAP-AMSP is represented with acidic (red), basic (blue), and hydrophobic (yellow) residues.

FIGURE 4.

FIGURE 4.

A, backbone 1H,15N hetero-NOE values of OSBPF in the unbound (white bar) and bound (black bar) state are shown. Uncertainties were obtained using Monte Carlo simulations. B, shown are changes of S2 values of VAP-AMSP upon complex formation with OSBPF. Δ_S_2 = _S_2(complex) − _S_2(free). The region between Δ_S_2 = −0.1 ∼ 0.1 is shadowed. C, composite chemical shift changes of VAP-AMSP and OSBPF upon complex formation are shown. Composite chemical shift changes were calculated using the equation Δppm = {(ΔδH)2 + (ΔδN/5)2}1/2, where ΔδH and ΔδN represent the chemical shift changes of 1H and 15N, respectively.

FIGURE 5.

FIGURE 5.

A, shown are cross-peaks of Cys-121 at a molar ratio of OSBPF to VAP-AMSP of 1:0. 44. In the upper panel, peaks are indicated by a cross and are labeled. The lower panel shows 1H cross-sections corresponding to lines a, b, and c in the upper panel. Numbers indicate corresponding peaks in the upper panel. B, shown is an overlay of 15N cross-sections of Gln-91 in a titration of VAP-AMSP with OSBPF. Molar ratios (VAP-AMSP:OSBPF) ranged from 1:0 (red) to 1:1.76 (blue). Peak tops at a molar ratio of 1:0.59 are shown by arrows. C, the upper panel shows an overlay of cross-peaks for Gln-74 at molar ratios of OSBPF of 0 (red), 0.59 (purple), and 1.76 (blue). The lower panel shows 1H cross-sections of peaks at each molar ratio. Corresponding peaks in the upper and lower panels are connected by dashed lines. D, shown is a comparison of cross-peaks for Cys-121 in titrations with OSBPF (WT) and the E356K mutant.

FIGURE 6.

FIGURE 6.

A, 1H,15N HSQC spectra of P56S (red) and WT (blue) VAP-AMSP are shown. In the boxed region, assignments of peaks of WT VAP-AMSP are indicated. B, 1H,15N HSQC spectra of P56S VAP-AMSP in the presence of OSBPF with a molar ratio of 1:2 (red) and in the absence of OSBPF (blue) are shown. C, DSC profiles of P56S (red) and WT (black) VAP-AMSP are shown.

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