Structural And Functional Characterizations Of The Multidrug Transporters Aq＿128 From Aquifex Aeolicus And PA2880 From Pseudomonas Aeruginosa

Multidrug resistance(MDR)transporters can confer the bacteria decreased susceptibility to antibiotics,although such decreases do not always result in clinical levels of antibiotic resistance.Many of these transporters are therefore of clinical relevance because they can render a bacterial infection untreatable by the agent(s)of choice.In order to understand the process of drug translocation mediated by MDR transporters,the multidrug and toxic compound extrusion(MATE)family member–Aq＿128 from Aquifex aeolicus and the proteobacterial antimicrobial compound efflux(PACE)family member–A2880 from Pesudomonas aeruginosa are investigated in this work.MATE transporters are widespread in all domains of life.Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H~+or Na~+to export xenobiotics across the membrane.Despite the availability of X-ray structures of several MATE transporters,a detailed understanding of the transport mechanism has remained elusive.In this work,we reported the crystal structure of Aq＿128 at 2.0??resolution.In the light of its phylogenetic placement outside the diversity of hitherto described MATE transporters and the lack of conserved acidic residues,this protein may represent a novel subfamily of the prokaryotic MATE transporters,which was proven by a phylogenetic analysis.Furthermore,the crystal structure and substrate docking results indicated that the substrate binding site is located in the N-bundle.The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis.We suggest that Aq＿128 may employ a different transport mechanism than previously proposed for the other types of MATE transporters.Our results provide structural insights into the transport mechanism of a novel MATE transporter,which further advance our global understanding of this important transporter family.The most recently identified PACE family transporters are mainly present in proteobacteria,and have not been found in archaea and eukaryotes to date.Investigation on a PACE family transporter,the PA2880 from Pseudomonas aeruginosa,was carried out in present work.Phylogenetic analysis of PACE family revealed that PA2880 and Ace I from Acinetobacter baumannii are classified into evolutionarily distinct clades,although both of them could transport chlorhexidine.We demonstrated that PA2880 exists as a dimer,and its dimerization is essential to proper function and independent on p H.Besides,PA2880 was proved to be a proton-dependent transporter,and the chlorhexidine/H~+antiport process is electrogenic according to the results of the electrogenicity experiments.The functional importance of several highly conserved residues was also investigated.These findings provide further insights into the functional features of PACE family transporter,facilitating the studies on their transport mechanisms.