Allosteric drug transport mechanism of multidrug transporter AcrB

Abstract

Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.

Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium

“…On the other hand, the St_AcrD I337A variant appears to confer a hyperactive phenotype (for aztreonam and temocillin). In contrast, the counterpart I337A substitution in Ec_AcrB resulted in a marked increased sensitivity towards β-lactam antibiotics, including oxacillin [ 25 , 31 ]. The overproduction of St_AcrD I337A (1.7-fold compared to St_AcrD WT, Figure S7 ) may possibly obscure its potential reduced ability to transport β-lactams.…”

“…This observation might possibly be in line with the reduction in hydrophobic binding pocket environment resulting in the enhancement of more hydrophilic substrate transport. Ala-substitutions made in the TM1/TM2 groove, proposed to be the initial binding site for carboxylated drugs in AcrB [ 25 , 31 ], conferred higher susceptibility to all four β-lactams tested ( Figure 2 , Figure S5 ). As an exception, I337A was shown to confer either wildtype-like or reduced susceptibility towards these substrates.…”

“…These channels displayed preferences for substrates on the basis of their physicochemical properties. [ 21 , 31 ].…”

Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR

“…Recently, the crystal structure of Cyanidioschyzon merolae , red algae, P-gp was determined in an outward-facing conformation with bound nucleotide, representing the first case that both inward and outward-facing conformations are determined for the eukaryotic homolog [ 77 ]. As for antiporter, the AcrAB has been extensively analyzed to unveil how trimeric AcrB transporter recognizes and eliminates various drugs in a functionally rotating mechanism based on the detailed three-dimensional structures [ 78 , 79 ]. Even a whole architecture of the AcrAB–TolC multidrug efflux pump that is composed of the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins, were experimentally visualized by Cryo-EM [ 79 , 80 ].…”

“…As for antiporter, the AcrAB has been extensively analyzed to unveil how trimeric AcrB transporter recognizes and eliminates various drugs in a functionally rotating mechanism based on the detailed three-dimensional structures [ 78 , 79 ]. Even a whole architecture of the AcrAB–TolC multidrug efflux pump that is composed of the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins, were experimentally visualized by Cryo-EM [ 79 , 80 ].…”