The Transmembrane Domain of a Bicomponent ABC Transporter Exhibits Channel-Forming Activity

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that expresses two unique forms of lipopolysaccharides (LPSs) on its bacterial surface, the A- and B-bands. The A-band polysaccharides (A-band PSs) are thought to be exported into the periplasm via a bicomponent ATP-binding cassette (ABC) transporter located within the inner membrane. This ABC protein complex consists of the transmembrane (TMD) Wzm and nucleotide-binding (NBD) Wzt domain proteins. Here, we were able to probe ~1.36 nS-average conductance openings of the Wzm-based protein complex when reconstituted into a lipid membrane buffered by a 200 mM KCl solution, demonstrating the large-conductance, channel-forming ability of the TMDs. In agreement with this finding, transmission electron microscopy (TEM) imaging revealed the ring-shaped structure of the transmembrane Wzm protein complex. As hypothesized, using liposomes we demonstrated that Wzm interacts with Wzt. Further, the Wzt polypeptide indeed hydrolyzed ATP, but exhibited a ~75% reduction in the ATPase activity when its Walker A domain was deleted. The distribution and average unitary conductance of the TMD Wzm protein complex were altered by the presence of the NBD Wzt protein, confirming the regulatory role of the latter polypeptide. To our knowledge, the large-conductance, channel-like activity of the Wzm protein complex, although often hypothesized, has not previously been demonstrated. These results constitute a platform for future structural, biophysical, and functional explorations of this bicomponent ABC transporter.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

“…The dilution ratio of the refolded protein within the bilayer chamber was ~ 1:1000. Therefore, the presence of detergent within the bilayer chamber did not affect the stability of the membrane; 45 ( D ) The unitary-conductance histograms of DDM-, OG-, and LysoFos-refolded FhuA ΔC/Δ5L. The electrical recordings were collected using 1M KCl, 10 mM potassium phosphate, pH 7.4.…”

“…40 μL of pure and denatured 6 × His + -tagged FhuA ΔC/Δ5L was 50-fold diluted into 29 mM DDM, 85 mM OG, or 16 mM LysoFos, containing 200 mM NaCl, 50 mM Tris.HCl, 1 mM EDTA, pH 8.0.The dilution ratio of the refolded protein within the bilayer chamber was ∼1:1000. Therefore, the presence of detergent within the bilayer chamber did not affect the stability of the membrane 45. (D) The unitary-conductance histograms of DDM-, OG-, and LysoFos-refolded FhuA ΔC/Δ5L.…”

Brucella melitensis Wzm/Wzt System: Changes in the Bacterial Envelope Lead to Improved Rev1Δwzm Vaccine Properties

“…In Brucella, this molecule is formed in the inner side of the cytoplasmic membrane. It is then translocated to the periplasm by an ATP-binding cassette (ABC) transport system that comprises two essential proteins, Wzt and Wzm, whereby the hydrophilic ATP-binding Wzt is coupled by a unique interface (Bi et al, 2018) to the transmembrane ring-shaped Wzm (Cuthbertson et al, 2007;Mohammad et al, 2016;Caffalette and Zimmer, 2021). This system is broadly conserved among gram-negative bacteria (Whitfield, 1995;Lerouge et al, 2001;Hug and Feldman, 2011;Caffalette et al, 2020), whereby Wzm is strongly conserved, while Wzt has a C-terminal domain (C-Wzt), with a unique structural element that determines the specificity of the O-PS transporter (Izquierdo et al, 2003;Cuthbertson et al, 2005Cuthbertson et al, , 2007.…”

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores

“…44, 45 The lipid bilayers were formed across a 100 µm-diameter aperture in a 25 µm-thick Teflon film (Goodfellow Corporation, Malvern, PA), which separated the cis and trans compartments. The aperture was pretreated with hexadecane (Sigma-Aldrich, St. Louis, MO) dissolved in highly purified pentane (Fisher HPLC grade, Fair Lawn, NJ) at a concentration of 10% (v/v).…”

“…All single-channel electrical recordings were conducted at room temperature using planar lipid bilayers with 1,2-diphytanoyl- sn -glycero-phosphocholine (Avanti Polar Lipids, Alabaster, AL), as previously described. , The lipid bilayers were formed across a 100 μm diameter aperture in a 25 μm thick Teflon film (Goodfellow Corp., Malvern, PA), which separated the cis and trans compartments. The aperture was pretreated with hexadecane (Sigma-Aldrich, St. Louis, MO) dissolved in highly purified pentane (high-performance liquid chromatography grade, Fisher, Fair Lawn, NJ) at a concentration of 10% (v/v).…”