Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated Protein A Revealed by NMR and Mutagenesis Studies

Kyoko Furuita,Jungoo Jee,Harumi Fukada,Masaki Mishima,Chojiro Kojima

doi:10.1074/jbc.m109.082602

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.

Highlights

We have successfully examined 1) the stoichiometry between VAP-AMSP and OSBPF and 2) the disorder property of OSBPF and 3) identified residues within and at the N-terminal side of the FFAT motif that contribute to the binding to VAP-AMSP
The apparent molecular mass value of the complex together with the ITC study indicates that the complex consists of one VAP-AMSP and one OSBPF
These results suggest the possibility that the complex is thought to be maintained by nonspecific interac- acidic patch of OSBPF, including Glu-356 of OSBPF, takes part tions considering the observations that the peaks in the middle in increasing the apparent binding affinity by contributing to of the titration have several peak tops and there is no clear the formation of an intermediate complex with VAP-AMSP

Summary

Introduction

Domain with OSBP Fragment—The solution structure of the complex of VAP-AMSP with OSBPF was determined. Amide proton resonances of Thr-46 and Val-54 of VAP-AMSP and Phe-360 of OSBPF shifted downfield upon complex formation, indicating formation of the respective hydrogen bonds.

Results

Conclusion