Molecular Interactions of Cephalosporins with the Deep Binding Pocket of the RND Transporter AcrB

Abstract

The drug/proton antiporter AcrB, part of the major efflux pump AcrABZ-TolC in Escherichia coli, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multidrug resistance phenotype. However, the molecular features differentiating between good and poor substrates of the pump have yet to be identified. In this work, we combined molecular docking with molecular dynamics simulations to study the interactions between AcrB and two representative cephalosporins, cefepime and ceftazidime (a good and poor substrate of AcrB, respectively). Our analysis revealed different binding preferences of the two compounds toward the subsites of the large deep binding pocket of AcrB. Cefepime, although less hydrophobic than ceftazidime, showed a higher affinity than ceftazidime for the so-called hydrophobic trap, a region known for binding inhibitors and substrates. This supports the hypothesis that surface complementarity between the molecule and AcrB, more than the intrinsic hydrophobicity of the antibiotic, is a feature required for the interaction within this region. Oppositely, the preference of ceftazidime for binding outside the hydrophobic trap might not be optimal for triggering allosteric conformational changes needed to the transporter to accomplish its function. Altogether, our findings could provide valuable information for the design of new antibiotics less susceptible to the efflux mechanism.

Molecular Interactions of Carbapenem Antibiotics with the Multidrug Efflux Transporter AcrB of Escherichia coli

“…This study contributes to explain the detailed interactions established by different carbapenem antibiotics with the DP T of AcrB in terms of their intrinsic physico-chemical properties. A retrospective analysis of the previous literature on other antimicrobial compounds [44] revealed the general applicability of our findings [35,55,[59][60][61].…”

The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux

“…6). Note that the contacts observed between FLE/ENR and the residues of the HP trap (and nearby hydrophobic residues) are present also in the available experimental structures of wild-type AcrB in complex with its substrates doxorubicin, minocycline, rhodamine-6G, and puromycin [29][30][31][32][33][34][35][36] .…”

“…We focused on the two main putative binding sites of AcrB known from structural studies 29,30 , namely the access pocket of the loose monomer (AP L ) and the distal pocket of the tight monomer (DP T ). Criteria adopted for the selection of the poses were: (i) the total number of docking poses in contact with at least 40% of the residues lining the two pockets 32,45 , (ii) the average values of the corresponding "binding affinities" (evaluated through the docking scoring function), and (iii) the frequency of contacts established between the compound and the residues lining the pockets.…”

“…Due to the substantial number of compounds to be screened, a reduced model of the AcrB protein not containing the transmembrane domain has been employed. The quality of the simplified model adopted has been validated in previous applications 32,33 . Each selected system was immersed in a box containing TIP3P water molecules 47 and an adequate number of K + counterions, in order to neutralize the negative net charge of the system.…”

“…However, the analysis of the docking poses provided multiple relevant configurations used as good starting points to perform two MD simulations for each FQ-AcrB complex. In particular, we employed a reduced model of AcrB not containing the transmembrane domain whose reliability has been thoroughly assessed in previous studies 32,33 . For each simulation considered, a cluster analysis was carried out on the MD trajectory followed by a binding free-energy evaluation through the Molecular Mechanics/Generalized Born Surface Area method.…”