Common recognition topology of mex transporters of Pseudomonas aeruginosa revealed by molecular modelling

Abstract

The secondary transporters of the resistance-nodulation-cell division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria like Pseudomonas aeruginosa. Among these RND transporters, MexB, MexF, and MexY, with partly overlapping specificities, have been implicated in pathogenicity. Only the structure of the former has been resolved experimentally, which together with the lack of data about the functional dynamics of the full set of transporters, limited a systematic investigation of the molecular determinants defining their peculiar and shared features. In a previous work (Ramaswamy et al., Front. Microbiol., 2018, 9, 1144), we compared at an atomistic level the two main putative recognition sites (named access and deep binding pockets) of MexB and MexY. In this work, we expand the comparison by performing extended molecular dynamics (MD) simulations of these transporters and the pathologically relevant transporter MexF. We employed a more realistic model of the inner phospholipid membrane of P. aeruginosa and more accurate force-fields. To elucidate structure/dynamics-activity relationships we performed physico-chemical analyses and mapped the binding propensities of several organic probes on all transporters. Our data revealed the presence, also in MexF, of a few multifunctional sites at locations equivalent to the access and deep binding pockets detected in MexB. Furthermore, we report for the first time about the multidrug binding abilities of two out of five gates of the channels deputed to peripheral (early) recognition of substrates. Overall, our findings help to define a common "recognition topology" characterizing Mex transporters, which can be exploited to optimize transport and inhibition propensities of antimicrobial compounds.

Keywords: Pseudomonas aeruginosa; RND efflux pumps; antibiotic resistance; molecular dynamics; molecular modeling; multidrug transporter.

FIGURE 1

(A) Side and top views of MexB X-ray crystal structure (PDB ID 3W9I) Loose (L; red), Tight (T; blue), and Open (O; green) protomers are shown in different colors. (B) The pre-MD structure of the MexF Open protomer. The funnel and inner transmembrane domains are shown in orange and blue, respectively. AP and DP binding sites are in green and red, respectively, and their geometrical centers are represented by spheres. PC1 and PN2 subdomains are in purple and skyblue, respectively. The G-loop is in yellow. All other periplasmic residues are shown in white. CH1, CH2, CH3, CH4, and CH5 domains are highlighted with cyan, seagreen, orange, yellow, and magenta, respectively, spheres having as centers the position of alpha carbons.

FIGURE 2

Electrostatic potential of (A) AP_L and (B) DP_T of MexB, MexF, and MexY. The electrostatic potential is plotted on the molecular surface representation of each binding pocket in the Pre-MD (left) and the most populated cluster (right) structures of MexB and its isoforms as seen from a periplasm. The color code is red to blue from negative (−10 k _b T/e) to positive (+10 k _b T/e) potentials, where k _b is the Boltzmann constant, T is the absolute temperature and e is the electron charge. PN2_L, PC1_L, PN2_T, and PC1_T domains of each RND transporter are in green and purple cartoons, respectively.

FIGURE 3

(A) Side and (B) top views of Loose (L) and Tight (T) protomers of the MexF pre-MD structure highlighting MFSs obtained with the FTMap server fragment-based mapping. Hydrogen bond donors, hydrogen bond acceptors, aliphatic, and aromatic organic probes are shown in celestial blue, blue, orange, and purple, respectively. The same color code of Figure 1 is applied to the protein and its channels. For the sake of clarity, only MFSs and CSs are shown. MFS_IG (IG: Interface/G-loop) is in proximity of the G-loop at the interface between AP and DP binding pockets. MFS_DP1 and MFS_DP2 denote the two different MFSs of the T protomer’s DP. (C) and (D) Zoomed views of MFSs located in L and T protomers, respectively, show sidechains of MexF residues interacting with each MFS, defined as those amino acids having at least one atom within 3.5 Å of any atom of the MFS small organic probes. Polar, negatively charged, positively charged, and hydrophobic sidechains are colored in green, red, blue, and white, respectively. For clarity, only MFSs of interest are shown in each panel, other adjacent MFSs and CSs are omitted.

FIGURE 4

L and T protomers of MexB, MexF, and MexY best representative structures highlighting MFSs obtained with the FTMap server fragment-based mapping. Hydrogen bond donors, hydrogen bond acceptors, aliphatic and aromatic organic probes are shown with the same color code in Figure 3. For clarity, AP and DP binding sites are represented by green and red spheres, respectively, centered at the geometric center of each domain. The same color code of Figure 1 is applied to the protein binding pockets, switch loop and channels. All the other protein residues of L and T protomers are shown in gray and white, respectively. The definition of MFS_AP, MFS_DP and MFS_IG is the same of Figure 3. MFS_CH2 and MFS_CH5 indicate MFSs in proximity of CH2 and CH5 channels, respectively. The sites not labeled as MFS are all CSs.