Both Acidic and Basic Amino Acids in an Amphitropic Enzyme, CTP:Phosphocholine Cytidylyltransferase, Dictate Its Selectivity for Anionic Membranes

Joanne E Johnson,Mingtang Xie,Laila M.R Singh,Robert Edge,Rosemary B Cornell

doi:10.1074/jbc.m206072200

Joanne E Johnson, Mingtang Xie + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m206072200

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Abstract

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.

Highlights

Proteins that interact reversibly with cell membrane lipids usually have selectivity for negatively charged phospholipids [1, 2]
In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding
The first, a strategy commonly used by amphitropic proteins, consists of strips of lysines flanking both sides of the nonpolar face of the amphipathic helix, which provide the electrostatic drive

Summary

Introduction

Proteins that interact reversibly with cell membrane lipids usually have selectivity for negatively charged phospholipids [1, 2]. Many proteins exhibit non-selectivity with respect to the anionic phospholipid by a simple electrostatic interaction between clusters of basic residues on the protein and negatively charged lipid head groups [6]. This binding affinity may be regulated by changes in membrane anionic lipid content, but more often by modification of the charge on the protein by a mechanism referred to as an “electrostatic switch” [7]. One of the interfacial strips is exclusively basic, and the other is a mixture of acidic and basic residues (Fig. 1, B and C)

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