Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy

Günnur Güler,Rebecca M Gärtner,Christine Ziegler,Werner Mäntele

doi:10.1074/jbc.m114.621979

Abstract

The Na(+)-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K(+) during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K(+) binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions.

Highlights

The secondary active betaine transporter BetP is the major defense system for the Gram-positive soil bacterium Corynebacterium glutamicum to counteract hyperosmotic stress
From the eight lipids observed in the trimeric structure, at least five lipids are located at functional important helices, mainly close to the cytoplasmic parts of the bundle helix transmembrane helice 3 (TMH3) and the scaffold helix TMH12 (Fig. 1D, lipids are colored similar to the helices with which they are interacting)
The osmo-regulated betaine transporter BetP is activated by increasing amounts of potassium ions and has shown a strong dependence of its activation profile on the amount of anionic lipids found in the surrounding membrane [5]

Summary

Introduction

The secondary active betaine transporter BetP is the major defense system for the Gram-positive soil bacterium Corynebacterium glutamicum to counteract hyperosmotic stress. From the eight lipids observed in the trimeric structure, at least five lipids are located at functional important helices, mainly close to the cytoplasmic parts of the bundle helix TMH3 and the scaffold helix TMH12 (Fig. 1D, lipids are colored similar to the helices with which they are interacting). These two helices are crucial in betaine transport [17] and often are referred to as gating helices. Positively charged residues are found in close proximity to the negatively charged PG head groups (Fig. 1C) the coordination is less specific than anticipated from the specific PG binding, which was tested previously by thin layer chromatography and mass spectrometry [16]

Methods

Results

Conclusion