Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump

Abstract

Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle. IMPORTANCE Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

“…1A, B, and C). This structure is very different from the cryo-EM structure of the Acinetobacter baumannii AdeB multidrug efflux pump (23), where the three apo-protomers have an identical conformation and form a symmetrical trimer. Each protomer of AdeB prefers a transient state in which the periplasmic cleft created by subdomains PC1 and PC2 is closed in conformation.…”

“…Additionally, each MtrD CR103 molecule by itself forms a PE binding site, which specifically anchors a lipid molecule (Fig. 5E), and this PE-binding site is conserved with that of the AdeB multidrug efflux pump (23).…”

Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from Salmonella

“…Optimisation of sample preparation allowed us to push the resolution to near 4 Å (in the periplasmic porter domains) of S TmAcrB, allowing confident placement of most side-chains. Our new S TmAcrB structure adds to only a few available recent native-lipid membrane structures of RND-transporters including the Acinetobacter baumannii AdeB [ 72 ] and Ec AcrB [ 62 ].…”

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

“…The purified AcrB protein was then concentrated to a final monomeric concentration of 10 μM in buffer containing 20 mM Na-HEPES (pH 7.5) in 0.05% DDM. Similar protein purification procedures have been used to elucidate structure-function of RND transporters, including AcrB [ 43 , 113 , 114 ], CusA [ 115 , 116 ], MtrD [ 117 , 118 ], CmeB [ 44 ], AdeB [ 119 ], HpnN [ 45 ] and MmpL3 [ 120 ]. We also used these protein purification protocols for in vitro substrate transport study via the CusA transporter [ 116 ] and in vitro functional dynamics measurement of the CmeB transporter [ 44 ], indicating that these purified membrane proteins are fully functional in vitro .…”