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. 2017 Aug 14;7(1):8075.
doi: 10.1038/s41598-017-08747-8.

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

Venkata Krishnan Ramaswamy  1 , Attilio V Vargiu  1 , Giuliano Malloci  1 , Jürg Dreier  2 , Paolo Ruggerone  3
Affiliations

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

Venkata Krishnan Ramaswamy et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Structure of the RND tripartite pump AcrAB-TolC. AcrAB-TolC pump is shown here with AcrB as the RND component, AcrA as the MFP and TolC as the OMP. In the inset, the general structure of an RND transporter is displayed, where the three main domains (TMD, PD, and FD) are indicated.
Figure 2
Figure 2
Proposed functional rotation mechanism of substrate extrusion by RND transporters. (a) Top view illustrating the different conformations assumed by AP, DP and Exit Gate (EG) during the three cycles of the functional rotation mechanism. The substrate (orange) is represented by van der Waals spheres and its pathway along the cycle is indicated by short black arrows. The key regions housing the substrate are coloured dull when binding to the substrate, bright otherwise. (b) Front view of the putative substrate transport pathway from AP to EG going through DP. The pathway is shown as thick tube, coloured in blue and magenta for the stages of the transport cycle associated to the Loose→Tight and Tight→Open conformational changes, respectively. The substrate is represented by sticks coloured green, red and iceblue when interacting with the AP, DP and EG (also coloured green, red and iceblue), respectively. The G-loop separating AP and DP is shown as a yellow cartoon.
Figure 3
Figure 3
Volume dynamics of AP in the Loose protomer of AcrB and AcrD. (Left Panels) Distribution of the volume of AP within the Loose protomer of AcrB (a) and AcrD (c), calculated for the 10 top cluster representatives extracted from equilibrium MD trajectories. Histograms refer to the values of the volume, lines to the relative population of the corresponding clusters. The volumes calculated for the pre-MD structures of AcrB and AcrD are shown as dashed line. (Right Panels) Porcupine plots representing collective motions along the first principal-component eigenvector for AP in AcrB (b) and AcrD (d) simulations shown as arrows (>2 Å) attached to Cα atoms indicating the magnitude of the corresponding eigenvalues. The loop lining the base of AP (‘bottom-loop’) showing large rearrangement in AcrB but not in AcrD are coloured yellow in both.
Figure 4
Figure 4
MLP and electrostatic potential of AP in the Loose protomer of AcrB and AcrD. MLP isosurfaces observed within 4 Å of AP in the Loose protomer of AcrB (blue) and AcrD (red) in pre-MD (a) and the representatives of the most populated cluster (b) as seen from the centre of the protomer. The hydrophobic/aromatic residues in the AP are shown as sticks in the structures. Isosurfaces at 0.75 (solid), 0.5 (dark transparent) and 0.25 (light transparent) are shown in blue (AcrB) or red (AcrD). The HP-trap and Vestibule sites are also labeled in the pre-MD structure of AcrB. The electrostatic potential plotted on the molecular surface representation of AP in the Acr proteins in the pre-MD (c) and the most populated cluster representative (d) as seen from the periplasmic front of the protomer. The colour code is red to blue from negative (−10 kbT/e) to positive (+10 kbT/e) potential, where kb is the Boltzmann constant, T is the absolute temperature and e is the electron charge.
Figure 5
Figure 5
Hydration of AP in the Loose protomer of AcrB and AcrD. (a) Comparison of RDF profiles of water oxygen atoms around AP (all atoms) in the Loose protomer of AcrB (red solid line) and AcrD (brown dash-dotted line) extracted from the equilibrium MD trajectories. (b) Comparison of SDF of waters within the AP of Loose protomer. The SDF was calculated over the configurations forming the most populated cluster of AcrB (left) and AcrD (right). The isosurfaces are shown at density isovalue of 6, meaning that the represented surfaces correspond to 6 times higher average number density of solvent molecules than bulk (see Subsection Hydration in Methods). The AP and DP are marked in green and red while the G-loop in yellow cartoon representations. The hydrophobic/aromatic residues of the pocket are shown as cyan sticks in the respective structures.
Figure 6
Figure 6
Volume dynamics of DP in the Tight protomer of AcrB and AcrD. (Left Panels) Volume distribution of DP in the Tight protomer of AcrB (a) and AcrD (c) over the simulation timescale. (Right Panels) Porcupine plots of the first principal-component eigenvector for DP in AcrB (b) and AcrD (d) simulations shown as arrows (>2 Å) attached to Cα atoms indicating the magnitude of the corresponding eigenvalues.
Figure 7
Figure 7
MLP and electrostatic potential of DP in the Tight protomer of AcrB and AcrD. See Fig. 4 for further details.
Figure 8
Figure 8
Hydration of DP and HP-trap in the Tight protomer of AcrB and AcrD. (a) Comparison of RDF profiles of water oxygen atoms around the DP (all atoms) and HP-trap (all atoms) in the Tight protomer of AcrB (red solid line) and the corresponding regions of AcrD (brown dash-dotted line). (b) Comparison of SDF for waters in the DP calculated over the configurations forming the most populated cluster of AcrB (left) and AcrD (right) illustrating the variation in the immediate environment of the hydrophobic residues. The position of the HP-trap in DP of AcrB is indicated by an arrow. See Fig. 5 for further details.
Figure 9
Figure 9
MFSs identified in the AP and DP of AcrB and AcrD. MFSs in the AP and DP of pre-MD (left panels) and the most populated cluster representative (right panel) structures of AcrB and AcrD. The binding modes of the different probes are shown as lines for hydrogen-bond donor (cyan), hydrogen-bond acceptor (violet) and aliphatic (yellow), and as CPK for aromatic (ochre) ligands. The AP and DP are marked in green and red, respectively, while the G-loop in yellow cartoon representations. (Note: The categorizing of MFSs here is arbitrary due to indistinct boundaries between the pockets. The sites not labelled as MFS here are all CSs).
Figure 10
Figure 10
Main conformational states of the Thr676-loop (bottom-loop) in the Loose protomer of AcrB (left panel) and of the corresponding Ser675-loop in AcrD (right panel). The conformation of the most populated clusters and the pre-MD structures are shown in red and grey cartoons, respectively. The conformations of the G-loop are also indicated with the same colour code.
Figure 11
Figure 11
Electrostatic funnel converging into AP in the Loose protomer of AcrB. Only the strongest field lines are shown and coloured red to blue from negative (−10 kbT/e) to positive (+10 kbT/e) potential (‘kb’ is the Boltzmann constant, ‘T’ is absolute temperature and ‘e’ is charge of an electron). AP is shown in green and the rest of the porter domain in white surface representation.
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