14 results found
Hohl M, Remm S, Eskandarian HA, et al., 2019, Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG, MOLECULAR MICROBIOLOGY, Vol: 111, Pages: 1263-1282, ISSN: 0950-382X
Arnold FM, Hohl M, Remm S, et al., 2018, A uniform cloning platform for mycobacterial genetics and protein production, SCIENTIFIC REPORTS, Vol: 8, ISSN: 2045-2322
Bärlocher K, Hutter CAJ, Swart AL, et al., 2017, Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5, Nature Communications, Vol: 8, ISSN: 2041-1723
Legionella pneumophila can cause Legionnaires’ disease and replicates intracellularly in adistinct Legionella-containing vacuole (LCV). LCV formation is a complex process thatinvolves a plethora of type IV-secreted effector proteins. The effector RidL binds the Vps29retromer subunit, blocks retrograde vesicle trafficking, and promotes intracellular bacterialreplication. Here, we reveal that the 29-kDa N-terminal domain of RidL (RidL2–281) adopts a“foot-like” fold comprising a protruding β-hairpin at its “heel”. The deletion of the β-hairpin,the exchange to Glu of Ile170 in the β-hairpin, or Leu152 in Vps29 abolishes the interaction ineukaryotic cells and in vitro. RidL2–281 or RidL displace the Rab7 GTPase-activating protein(GAP) TBC1D5 from the retromer and LCVs, respectively, and TBC1D5 promotes theintracellular growth of L. pneumophila. Thus, the hydrophobic β-hairpin of RidL is critical forbinding of the L. pneumophila effector to the Vps29 retromer subunit and displacement of theregulator TBC1D5.
Hürlimann LM, Hohl M, Seeger MA, 2017, Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD., FEBS J, Vol: 284, Pages: 1672-1687
Many heterodimeric ATP-binding cassette (ABC) exporters evolved asymmetric ATP-binding sites containing a degenerate site incapable of ATP hydrolysis due to noncanonical substitutions in conserved sequence motifs. Recent studies revealed that nucleotide binding to the degenerate site stabilizes contacts between the nucleotide-binding domains (NBDs) of the inward-facing transporter and regulates ATP hydrolysis at the consensus site via allosteric coupling mediated by the D-loops. However, it is unclear whether nucleotide binding to the degenerate site is strictly required for substrate transport. In this study, we examined the functional consequences of a systematic set of mutations introduced at the degenerate and consensus site of the multidrug efflux pump EfrCD of Enterococcus faecalis. Mutating motifs which differ among the two ATP-binding sites (Walker B, switch loop, and ABC signature) or which are involved in interdomain communication (D-loop and Q-loop) led to asymmetric results in the functional assays and were better tolerated at the degenerate site. This highlights the importance of the degenerate site to allosterically regulate the events at the consensus site. Mutating invariant motifs involved in ATP binding and NBD closure (A-loop and Walker A) resulted in equally reduced transport activities, regardless at which ATP-binding site they were introduced. In contrast to previously investigated heterodimeric ABC exporters, mutation of the degenerate site Walker A lysine completely inactivated ATPase activity and substrate transport, indicating that ATP binding to the degenerate site is essential for EfrCD. This study provides novel insights into the split tasks of asymmetric ATP-binding sites of heterodimeric ABC exporters.
Timachi MH, Hutter CAJ, Hohl M, et al., 2017, Exploring conformational equilibria of a heterodimeric ABC transporter, eLife, Vol: 6, ISSN: 2050-084X
ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.
Hürlimann LM, Corradi V, Hohl M, et al., 2016, The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis., Antimicrobial Agents and Chemotherapy, Vol: 60, Pages: 5400-5411, ISSN: 0066-4804
Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.
Seeger MA, Bordignon E, Hohl M, 2015, ABC Exporters from a Structural Perspective, ABC Transporters - 40 Years on, Publisher: Springer, ISBN: 9783319234762
In Part I, A Structure-Function Perspective, Chapters 'ABC Importers' and 'Bacterial ABC Multidrug Exporters: From Shared Proteins Motifs and Features to Diversity in Molecular Mechanisms' are devoted to an overview of the two major types ...
Bukowska MA, Hohl M, Geertsma ER, et al., 2015, A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter., Biochemistry, Vol: 54, Pages: 3086-3099
ABC exporters are ubiquitous multidomain transport proteins that couple ATP hydrolysis at a pair of nucleotide binding domains to substrate transport across the lipid bilayer mediated by two transmembrane domains. Recently, the crystal structure of the heterodimeric ABC exporter TM287/288 was determined. One of its asymmetric ATP binding sites is called the degenerate site; it binds nucleotides tightly but is impaired in terms of ATP hydrolysis. Here we report the crystal structures of both isolated motor domains of TM287/288. Unexpectedly, structural elements constituting the degenerate ATP binding site are disordered in these crystals and become structured only in the context of the full-length transporter. In addition, hydrogen bonding patterns of key residues, including those of the catalytically important Walker B and the switch loop motifs, are fundamentally different in the solitary NBDs compared to those in the intact transport protein. The structures reveal crucial interdomain contacts that need to be established for the proper assembly of the functional transporter complex.
Marino J, Hohl M, Seeger MA, et al., 2015, Bicistronic mRNAs to enhance membrane protein overexpression., J Mol Biol, Vol: 427, Pages: 943-954
Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy, we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.
Hohl M, Hürlimann LM, Böhm S, et al., 2014, Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter., Proc Natl Acad Sci U S A, Vol: 111, Pages: 11025-11030
ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.
Hohl M, Grütter MG, Seeger MA, 2012, X-Ray Structure of a Heterodimeric ABC Transporter Crystallized in its Inward-Facing Conformation., Chimia (Aarau), Vol: 66, ISSN: 0009-4293
Hohl M, Briand C, Grütter MG, et al., 2012, Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation., Nat Struct Mol Biol, Vol: 19, Pages: 395-402
ATP-binding cassette (ABC) transporters shuttle a wide variety of molecules across cell membranes by alternating between inward- and outward-facing conformations, harnessing the energy of ATP binding and hydrolysis at their nucleotide binding domains (NBDs). Here we present the 2.9-Å crystal structure of the heterodimeric ABC transporter TM287-TM288 (TM287/288) from Thermotoga maritima in its inward-facing state. In contrast to previous studies, we found that the NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP hydrolysis sites. We observed AMP-PNP binding to the degenerate catalytic site, which deviates from the consensus sequence in the same positions as the eukaryotic homologs CFTR and TAP1-TAP2 (TAP1/2). The TM287/288 structure provides unprecedented insights into the mechanism of heterodimeric ABC exporters and will enable future studies on this large transporter superfamily.
Seeger MA, Mittal A, Velamakanni S, et al., 2012, Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders., PLoS One, Vol: 7
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.
Zobi F, Hohl M, Zimmermann I, et al., 2004, Binding of 9-methylguanine to [cis-Ru(2,2'-bpy)2]2+: First X-ray structure of a cis-bis purine complex of ruthenium., Inorg Chem, Vol: 43, Pages: 2771-2772, ISSN: 0020-1669
Reaction of [cis-Ru(2,2'-bpy)2(O3SCF3)2] (1) with 9-methylguanine (9-MeG) affords the cis-[Ru(2,2'-bpy)2(9-MeG)2]2+ complex (2) in good yield. Two bases bind to the metal center via the N7 atoms. X-ray structure analysis of 2(SO3CF3)2 (monoclinic, P2(1)/n, a = 12.5159(6) A, b = 20.0904(13) A, c = 17.1202(9) A, beta = 98.981(6) degrees, V = 4252.1(4) A(3), Z = 4) reveals that the two bases are in a head-to-tail (HT) orientation with base-base dihedral angle of 60.4 degrees. NMR studies confirm that the complex is stable in water for hours, and no evidence for guanine substitution by solvent molecules was found.
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