Cryo-EM as a tool to study bacterial efflux systems and the membrane proteome

Abstract

Antibiotic resistance is an emerging threat to global health. Current treatment regimens for these types of bacterial infections are becoming increasingly inadequate. Thus, new innovative technologies are needed to help identify and characterize novel drugs and drug targets which are critical in order to combat multidrug-resistant bacterial strains. Bacterial efflux systems have emerged as an attractive target for drug design, as blocking their export function significantly increases the potency of administered antibiotics. However, in order to develop potent and tolerable efflux pump inhibitors with high efficacy, detailed structural information is required for both the apo- and substrate-bound forms of these membrane proteins. The emergence of cryo-electron microscopy (cryo-EM) has greatly advanced the field of membrane protein structural biology. It has significantly enhanced the ability to solve large multi-protein complexes as well as extract meaningful data from a heterogeneous sample, such as identification of several assembly states of the bacterial ribosome, from a single data set. This technique can be expanded to solve the structures of substrate-bound efflux pumps and entire efflux systems from previously unusable membrane protein sample preparations. Subsequently, cryo-EM combined with other biophysical techniques has the potential to markedly advance the field of membrane protein structural biology. The ability to discern complete transport machineries, enzymatic signal transduction pathways, and other membrane-associated complexes will help us fully understand the complexities of the membrane proteome.

Keywords: Antibiotic resistance; RND-type transporters; cryo-electron microscopy.

Figure 1.. General structure of trimeric RND efflux systems.

(A) The components of a tripartite efflux system (adapted from Protein Data Bank ID 5O66) visualized in side view with the inner membrane pump (IMP, blue), membrane fusion protein (MFP, green) and outer membrane protein (OMP, purple). Pictured is the IMP:MFP:OMP subunit ratio of 3:6:3, which is the most common assembly pattern. The outer membrane (OM) and inner membrane (IM) are designated by dashed lines. (B) A magnified view of one subunit of an RND-type inner membrane pump. Periplasmic subunits are designated DC (yellow), DN (red), PN1 (light blue), PN2 (purple), PC1 (orange), and PC2 (green). The transmembrane (TM) and membrane-associated helices are designated blue. RND, resistance–nodulation–cell division.

Figure 2.. Antibiotic-bound cryo-EM structure of the Neisseria gonorrhoeae RND-type inner membrane pump, MtrD (adapted from Protein Data Bank ID 6VKS).

(A) α-helices (blue), β-sheets (wheat), and loops (gray) depict the overall secondary structure of MtrD. A hydrolyzed, decarboxylated ampicillin molecule (green) is bound deep within the cavity formed by the orientation of the periplasmic domains PC1, PC2, PN1, and PN2. The inner membrane–periplasm lipid boundary is represented by a dashed line. (B) A magnified view of the ampicillin-binding region. Important amino acid side chains involved in substrate recognition/stabilization are shown in orange. Amp, hydrolyzed, decarboxylated ampicillin; cryo-EM, cryo-electron microscopy; RND, resistance–nodulation–cell division.

Figure 3.. Ribosomal structures determined by single particle cryo-EM.

(A) Two-dimensional class averages obtained from a single cryo-EM experiment. Ribosomes were isolated from the Acinetobacter baumannii bacterium and flash-frozen onto a cryogenic grid. Shown are three separate classes that differentiate the 70S complex (red squares) from the individual 50S (blue circles) and the 30S (yellow hexagons) subunits. (B) Further computational sorting and analysis revealed three separate states of the intact 70S ribosome: tRNA bound at the P-site (green spheres), the E-site (red spheres), or empty. cryo-EM, cryo-electron microscopy.