Molecular Rationale behind the Differential Substrate Specificity of Bacterial RND Multi-Drug Transporters

Abstract

Resistance-Nodulation-cell Division (RND) transporters AcrB and AcrD of Escherichia coli expel a wide range of substrates out of the cell in conjunction with AcrA and TolC, contributing to the onset of bacterial multidrug resistance. Despite sharing an overall sequence identity of ~66% (similarity ~80%), these RND transporters feature distinct substrate specificity patterns whose underlying basis remains elusive. We performed exhaustive comparative analyses of the putative substrate binding pockets considering crystal structures, homology models and conformations extracted from multi-copy μs-long molecular dynamics simulations of both AcrB and AcrD. The impact of physicochemical and topographical properties (volume, shape, lipophilicity, electrostatic potential, hydration and distribution of multi-functional sites) within the pockets on their substrate specificities was quantitatively assessed. Differences in the lipophilic and electrostatic potentials among the pockets were identified. In particular, the deep pocket of AcrB showed the largest lipophilicity convincingly pointing out its possible role as a lipophilicity-based selectivity filter. Furthermore, we identified dynamic features (not inferable from sequence analysis or static structures) such as different flexibilities of specific protein loops that could potentially influence the substrate recognition and transport profile. Our findings can be valuable for drawing structure (dynamics)-activity relationship to be employed in drug design.

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

“…All of the 52 complexes selected from docking runs were subjected to all-atom molecular dynamics (MD) simulations (each of 1 μs in length) performed with the AMBER18 package 55 . Protomer-specific protonation states of AcrB were adopted following previous work 56 : residues E346 and D924 were protonated only in the L and T protomers, while residues D407, D408, and D566 were protonated only in the O protomer, of AcrB. The topology and the initial coordinate files were created using the LEaP module of the AMBER18 package.…”

“…Each system was first subjected to a multi-step structural relaxation via a combination of steepest descent and conjugate gradient methods using the pmemd program implemented in AMBER18, as described in previous publications 4,29,56 . The systems were then heated from 0 to 310 K in two subsequent MD simulations: i) from 0 to 100 K in 1 ns under constant-volume conditions and with harmonic restraints (k = 1 kcal·mol -1 ·Å -2 ) on the heavy atoms of both the protein and the lipids; ii) from 100 to 310 K in 5 ns under constant pressure (set to a value of 1 atm) and with restraints on the heavy atoms of the protein and on the z coordinates of the phosphorous atoms of the lipids to allow membrane rearrangement during heating.…”

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance

“…Genomes of Gram-negative bacteria encode multiple RND-transporters which pair with a number of PAPs forming a variety of efflux systems with different substrate profiles and distinct cellular roles [8,[10][11][12][13][14][15]. Salmonella has five RND transporters (AcrB, AcrD, AcrF, MdtB/ C and MdsB) while E. coli has six and Pseudomonas aeruginosa has more than ten.…”

Colicin E1 opens its hinge to plug TolC

“…TolC is the outer membrane component of the acridine efflux pump (along with AcrA in the periplasm and AcrB in the inner membrane) (6), which extrudes multiple classes of antibiotics such as erythromycin, chloramphenicol, tetracycline, doxorubicin, and acriflavine (7, 8), as well as other compounds such as bile salts and detergents (9). Deletion of TolC has been shown to render E. coli vulnerable to a wide variety of antibiotics (10).…”