Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.

ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters

Drug‐dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae

Funder: Croucher Foundation; Id: http://dx.doi.org/10.13039/501100001692The bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug-mimetic azido-ethidium. Surprisingly, our analyses of azido-ethidium-labelled PatAB peptides point to ethidium binding in the PatA nucleotide-binding domain, with the azido moiety crosslinked to residue Q521 in the H-like loop of the degenerate nucleotide-binding site. Investigation into this compound and residue’s role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium-dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium-like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non-competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide-binding domains

Study of W boson production in PbPb and pp collisions at sqrt(s[NN]) = 2.76 TeV

A measurement is presented of W-boson production in PbPb collisions carried out at a nucleon-nucleon (NN) centre-of-mass energy sqrt(s[NN]) of 2.76 TeV at the LHC using the CMS detector. In data corresponding to an integrated luminosity of 7.3 inverse microbarns, the number of W to mu mu-neutrino decays is extracted in the region of muon pseudorapidity abs(eta[mu])<2.1 and transverse momentum pt[mu]>25 GeV. Yields of muons found per unit of pseudorapidity correspond to (159 +/- 10 (stat.) +/- 12 (syst.)) 10E-8 W(plus) and (154 +/- 10 (stat.) +/- 12 (syst.)) 10E-8 W(minus) bosons per minimum-bias PbPb collision. The dependence of W production on the centrality of PbPb collisions is consistent with a scaling of the yield by the number of incoherent NN collisions. The yield of W bosons is also studied in a sample of pp interactions at sqrt(s)= 2.76 TeV corresponding to an integrated luminosity of 231 inverse nanobarns. The individual W(plus) and W(minus) yields in PbPb and pp collisions are found to agree, once the neutron and proton content in Pb nuclei is taken into account. Likewise, the difference observed in the dependence of the positive and negative muon production on pseudorapidity is consistent with next-to-leading order perturbative QCD calculations.Comment: Submitted to Physics Letters

DARPin expression does not significantly alter expression of LmrCD proteins.

<p>(<b>A</b>, <b>B</b>) A V5-tag was introduced in frame at the 5′-end of genomic <i>lmrD</i> in <i>L. lactis</i> (denoted <i>L. lactis NZ9000 lmrD_V5</i>). Plasmid-encoded DARPin activators or the control DARPin E3_5* were expressed in <i>L. lactis NZ9000 lmrD_V5</i> in the presence and absence of daunomycin (14 µM for DARPin_Act3 and E3_5* and 28 µM for DARPin_Act1 and DARPin_Act2, respectively). The expression levels of genomic LmrD_V5 were then quantified by comparing the Western blot signal obtained using an anti-V5 antibody (A) with total protein detected by SYPRO ruby staining (B). (<b>C</b>) The relative amounts of LmrD_V5 expression were quantified by densitometry. Each bar represents the average of three independent data points (n = 3) of which one data point is shown in (A) and (B).</p