Molecular rationale for the impairment of the MexAB-OprM efflux pump by a single mutation in MexA

Abstract

Efflux pumps of the Resistance-Nodulation-cell Division (RND) superfamily contribute to intrinsic and acquired resistance in Gram-negative pathogens by expelling chemically unrelated antibiotics with high efficiency. They are tripartite systems constituted by an inner-membrane-anchored transporter, an outer membrane factor protein, and a membrane fusion protein. Multimerization of the membrane fusion protein is an essential prerequisite for full functionality of these efflux pumps. In this work, we employed complementary computational techniques to investigate the stability of a dimeric unit of MexA (the membrane fusion protein of the MexAB-OprM RND efflux pump of Pseudomonas aeruginosa), and to provide a molecular rationale for the effect of the G72S substitution, which affects MexAB-OprM functionality by impairing the assembly of MexA. Our findings indicate that: i) dimers of this protein are stable in multiple µs-long molecular dynamics simulations; ii) the mutation drastically alters the conformational equilibrium of MexA, favouring a collapsed conformation that is unlikely to form dimers or higher order assemblies. Unveiling the mechanistic aspects underlying large conformational distortions induced by minor sequence changes is informative to efforts at interfering with the activity of this elusive bacterial weapon. In this respect, our work further confirms how molecular simulations can give important contribution and useful insights to characterize the mechanism of highly complex biological systems.

Keywords: Bacterial resistance; Molecular docking; Molecular dynamics; Protein structure and dynamics; RND efflux pumps.

Graphical abstract

Fig. 1

X-ray crystal structure of the MexA protein (PDB ID: 2V4D , chain L, residues 36 to 362 according the full Uniprot sequence numbering employed by Nehme et al. , or 13 to 339 according to the sequence numbering employed in the X-ray crystal structure numbering. The front view of the protein is shown on the left. The four domains of the protein are highlighted with different colors: α-hairpin (red; residues 96–158), lipoyl (green; residues 61–95, 159–194), β-barrel (blue; residues 50–60, 195–285) and Membrane Proximal (MP, mauve; residues 36–49, 286–362). The black sphere in the lipoyl domain identifies the mutation G72S considered in this work. A Zooming on the lipoyl domain is repoerted on the right side of the picture.

Fig. 2

Conformational dynamics of the WT and G72S variant of MexA. A) Conformations of the top five structural clusters extracted from the equilibrium trajectories of MexA_WT1 (left) and MexA_WT2 (right) compared to the X-ray structure of the protein (chain L from PDB ID 2V4D , coloured as in Fig. 1). The clusters are shown as grey ribbons, the top one being solid and the 2nd to 5th increasingly transparent. B) Same as in A for MexA_G72S1 (left) and MexA_G72S2 (right). C) Profiles of the RMSD (upper panel), radius of gyration (RoG, middle), and RMSF (lower) extracted from the simulations of WT and G72S variant of MexA. RMSD and RMSF were calculated after alignment of the lipoyl and β-domains of the protein. G72S1/2₁ and G72S1/2₂ refer to the RMSF profiles calculated respectively before and after protein collapse.

Fig. 3

Main rearrangements occurring in the G72S variant of MexA after ∼ 1.5 μs in two independent MD simulations. The structure evolved from an elongated shape (left) into a more compact conformation (right), in which the α-hairpin and the MP domains bend towards the β-barrel domain. In the right image the two conformations were aligned by matching their lipoyl and β-barrel domains in order to highlight the rotations of the peripheral domains (indicated by black arrows).

Fig. 4

A) Conformations of the twelve top ranked docking poses of MexA_WT-MexA_WT (left). The reference X-ray structure (2V4D) is shown as black ribbons, while the poses are shown in semi-transparent dark (chain L) and light (chain M) ribbons coloured from red to white to blue according to their docking score. The arrow on the right points to the conformations assumed by these poses at the end of MD simulations of 1 μs in length. B) Conformations of the twelve top ranked docking poses of MexA_G72S-MexA_G72S. C) Fraction of native contacts f_NC detected in the twelve top ranked docking poses for ${\text{MexA}}_{\text{2dock}}^{\text{WT}}$ and ${\text{MexA}}_{\text{2dock}}^{\text{G72S}}$. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)