Poster abstracts
Poster number 9 submitted by Brent Simpson
Complex formation of the ABC transporter that couples ATP hydrolysis to LPS transport in Escherichia coli
Brent W Simpson (Department of Microbiology, OSU), Matt Orabella (Department of Microbiology, OSU), Rebecca Davis (Department of Microbiology, OSU), Natividad Ruiz (Department of Microbiology, OSU)
Abstract:
The defining trait of Gram-negative bacteria is an outer membrane which typically contains lipopolysaccharide (LPS). When present LPS is located exclusively on the cell surface and its many sugar groups and charges form a dense hydrophilic layer before the hydrophobic membrane environment. These unique characteristics provide Gram-negative bacteria with intrinsic resistance to many antibiotics. E. coli requires a seven-protein complex, LptAB2CDEFG, to transport LPS from its site of synthesis, the inner membrane, to its site of function, the outer membrane. An unusual ABC transporter, LptB2FG, utilizes ATP to power LPS transport. To perform this function LptB hydrolyses cytoplasmic ATP and must transmit conformational information to transmembrane proteins LptFG. LptFG must then directly or indirectly extract LPS from the periplasmic side of the inner membrane. Here we explore the interacting domains and residues that perform this energy coupling between LptB2 and LptFG. We hypothesized that a structural groove in LptB, previously identified in crystal structures, interacts with coupling helices in LptFG that we have identified using bioinformatics. Structure-function analysis of both the groove of LptB and the putative coupling helices of LptFG revealed residues essential for LPS transport. We further demonstrated direct interactions between the groove of LptB and LptFG using photo-crosslinkable, unnatural amino acid substitutions at specific sites in LptB. To dissect the role of the putative coupling helices of LptFG, we selected for spontaneous suppressors of a double mutant (lptFE84A lptGE88A) with defects in the coupling helices. We isolated a suppressor with a single amino acid substitution in the groove of LptB, establishing a functional connection between the coupling helices and groove. This study provides initial insights into the process of coupling a cytoplasmic energy source to LPS transport across the periplasm and outer membrane.
Keywords: ATP-binding cassette transporter, lipopolysaccharide, cell envelope biogenesis