Abstract
Secondary multidrug transporters of the resistance-nodulation-cell division (RND) superfamily contribute crucially to antibiotic resistance in Gram-negative bacteria. Compared to the most studied transporter AcrB of Escherichia coli, little is known about the molecular determinants of distinct polyspecificities of the most important RND transporters MexB and MexY of Pseudomonas aeruginosa. In an effort to add knowledge on this topic, we performed an exhaustive atomic-level comparison of the main putative recognition sites (access and deep binding pockets) in these two Mex transporters. We identified an underlying link between some structural, chemical and dynamical features of the binding pockets and the physicochemical nature of the corresponding substrates recognized by either one or both pumps. In particular, mosaic-like lipophilic and electrostatic surfaces of the binding pockets provide for both proteins several multifunctional sites for diffuse binding of diverse substrates. Specific lipophilicity signatures of the weakly conserved deep pocket suggest a key role of this site as a selectivity filter as in Acr transporters. Finally, the different dynamics of the bottom-loop in MexB and MexY support its possible role in binding of large substrates. Our work represents the first comparative study of the major RND transporters in P. aeruginosa and also the first structure-based study of MexY, for which no experimental structure is available yet.
Highlights
Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections worldwide due to the emergence and spread of multi, extensive, and pan-drug resistant isolates susceptible to very few antimicrobial agents (Fischbach and Walsh, 2009; Poole, 2011)
MexB resembles AcrB with a jellyfishlike structural topology formed by an asymmetric trimer with each protomer comprising three domains (Ruggerone et al, 2013) (Figure 1A): (i) a trans-membrane domain (TMD) of 12 α-helices embedded in the inner membrane (IM), where the chemical-to-mechanical energy conversion takes place; (ii) a pore domain (PD) located in the periplasm, where substrate recruitment and transport occur; and (iii) a periplasmic funnel domain (FD), which connects the resistance-nodulation-cell division (RND) transporter to the outer membrane protein (OMP) via the assembly of membrane fusion proteins (MFPs) (Symmons et al, 2015) in the constituted pump
Since bacteria respond to adverse environmental stress by altering their genetic makeup, we first analyzed the sequences of MexB and MexY from all available bacterial strains of P. aeruginosa
Summary
Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections worldwide due to the emergence and spread of multi, extensive, and pan-drug resistant isolates susceptible to very few antimicrobial agents (Fischbach and Walsh, 2009; Poole, 2011). Several RND type efflux systems have been identified in P. aeruginosa PAO1 (Webber and Piddock, 2003; Poole, 2005; Lister et al, 2009; Zechini and Versace, 2009; Fernández and Hancock, 2012; Blair et al, 2014; Delmar et al, 2014; Sun et al, 2014), with the most significant for multidrug resistance being MexAB-OprM (Poole et al, 1993; Gotoh et al, 1995) and MexXYOprM (Aires et al, 1999; Mine et al, 1999; Westbrock-Wadman et al, 1999) These two machineries contribute additively to the resistance to common substrate antibiotics (Lee et al, 2000; Llanes et al, 2004); their different specificities (viz. MexB for β-lactams and MexY for aminoglycosides) drastically reduce the susceptibility of infectious strains to numerous classes of antibiotics (Llanes et al, 2004). The pockets are separated by a G-rich (a.k.a. switch) loop whose flexibility has been shown to be important for the transport of high-molecular mass compounds (Nakashima et al, 2011; Eicher et al, 2012)
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