Abstract
The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.
Highlights
For many pathogens, carbohydrates are critical carbon sources for the production of energy and essential biomolecules, which are required for a wide range of cellular processes
To establish whether the LpqY-SugABC transporter is specific for trehalose or is instead promiscuous for other carbohydrates, a panel of monosaccharides and disaccharides (10 mM) were screened for their ability to stabilize the melting temperature (Tm) of the Mycobacterium thermoresistible (Mtr) LpqY substrate binding domain
Trehalose is a key component of the mycobacterial cell envelope, and “free” trehalose, released from the trehalose-containing glycolipids, is recovered by the LpqY-SugABC ATP-binding cassette (ABC) transporter [6]
Summary
Carbohydrates are critical carbon sources for the production of energy and essential biomolecules, which are required for a wide range of cellular processes. These data establish that the Mtr LpqY substrate binding protein is highly specific for trehalose. The MST was used to determine the binding affinities of each site-directed Mtr LpqY mutant protein with trehalose.
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