Role of the Transmembrane Domain in the Stability of TrwB, an Integral Protein Involved in Bacterial Conjugation

Itsaso Hormaeche,Ibón Iloro,José L.R Arrondo,Félix M Goñi,Fernando De La Cruz,Itziar Alkorta

doi:10.1074/jbc.m310422200

Abstract

TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed bacterial conjugation. The protein consists of a cytosolic domain, which contains an ATP-binding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwBDeltaN70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwBDeltaN70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwBDeltaN70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.

Highlights

The purification and study of integral membrane proteins present serious problems, derived from the requirements of a partly aqueous, partly hydrophobic environment for the maintenance of a native-like conformation and activity by those proteins [1,2,3,4]
These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane
As a part of an investigation on the conjugation proteins encoded by plasmid R388, the study of TrwB was undertaken in our laboratories

Summary

Introduction

The purification and study of integral membrane proteins present serious problems, derived from the requirements of a partly aqueous, partly hydrophobic environment for the maintenance of a native-like conformation and activity by those proteins [1,2,3,4]. Because of the difficulty in handling the entire membrane protein, a soluble form of TrwB lacking the N-terminal transmembrane segments (TrwB⌬N70) was designed, purified, and partially characterized [13] This mutant protein shed some light on the role of TrwB in the conjugation process [13, 15, 16]. The intact protein binds ATP with significantly higher affinity than the truncated soluble form Availability of both the native protein (TrwB) and the truncated mutant lacking the transmembrane domain (TrwB⌬N70) prompted us to compare the stability of both protein forms. Truncated TrwB was found to be stabilized by detergent, but even in this case stability was lower than for whole TrwB These results provide direct evidence for the role of the transmembrane anchor in maintaining the functional conformation of an extramembranous domain in an integral membrane protein

Methods

Results

Conclusion