In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.
- Award ID(s):
- 1845012
- PAR ID:
- 10389101
- Date Published:
- Journal Name:
- eLife
- Volume:
- 11
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
more » « less
Abstract Cationic biocides play a crucial role in the disinfection of domestic and healthcare surfaces. Due to the rise of bacterial resistance towards common cationic disinfectants like quaternary ammonium compounds (QACs), the development of novel actives is necessary for effective infection prevention and control. Toward this end, a series of 15 chimeric biscationic amphiphilic compounds, bearing both ammonium and phosphonium residues, were prepared to probe the structure and efficacy of mixed cationic ammonium‐phosphonium structures. Compounds were obtained in two steps and good yields, with straightforward and chromatography‐free purifications. Antibacterial activity evaluation of these compounds against a panel of seven bacterial strains, including two MRSA strains as well as opportunistic pathogen
A. baumannii , were encouraging, as low micromolar inhibitory activity was observed for multiple structures. Alkyl chain length on the ammonium group was, as expected, a major determinant of bioactivity. In addition, high therapeutic indexes (up to 125‐fold) for triphenyl phosphonium‐bearing amphiphiles were observed when comparing antimicrobial activity to mammalian cell lysis activity. -
more » « less
Abstract Inspired by the incorporation of metallocene functionalities into a variety of bioactive structures, particularly antimicrobial peptides, we endeavored to broaden the structural variety of quaternary ammonium compounds (QACs) by the incorporation of the ferrocene moiety. Accordingly, 23 ferrocene‐containing mono‐ and bisQACs were prepared in high yields and tested for activity against a variety of bacteria, including Gram‐negative strains and a panel of clinically isolated MRSA strains. Ferrocene QACs were shown to be effective antiseptics with some displaying single‐digit micromolar activity against all bacteria tested, demonstrating yet another step in the expansion of structural variety of antiseptic QACs.
-
Antivirulence strategy has been explored as an alternative to traditional antibiotic development. The bacterial type IV pilus is a virulence factor involved in host invasion and colonization in many antibiotic resistant pathogens. The PilB ATPase hydrolyzes ATP to drive the assembly of the pilus filament from pilin subunits. We evaluated Chloracidobacterium thermophilum PilB (CtPilB) as a model for structure-based virtual screening by molecular docking and molecular dynamics (MD) simulations. A hexameric structure of CtPilB was generated through homology modeling based on an existing crystal structure of a PilB from Geobacter metallireducens. Four representative structures were obtained from molecular dynamics simulations to examine the conformational plasticity of PilB and improve docking analyses by ensemble docking. Structural analyses after 1 μs of simulation revealed conformational changes in individual PilB subunits are dependent on ligand presence. Further, ensemble virtual screening of a library of 4234 compounds retrieved from the ZINC15 database identified five promising PilB inhibitors. Molecular docking and binding analyses using the four representative structures from MD simulations revealed that top-ranked compounds interact with multiple Walker A residues, one Asp-box residue, and one arginine finger, indicating these are key residues in inhibitor binding within the ATP binding pocket. The use of multiple conformations in molecular screening can provide greater insight into compound flexibility within receptor sites and better inform future drug development for therapeutics targeting the type IV pilus assembly ATPase.more » « less
-
Cleavage of aromatic carbon–chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (Chd T ), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. Chd T forms a “head-to-tail” homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His 117 , His 257 , Asp 116 , Asn 216 , and a water/hydroxide as ligands. A conserved His residue, His 114 , is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π–π interaction with Trp 227 . On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.76766
