scholarly journals Defining the RND-binding residues of AcrA

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Ilyas Alav ◽  
Ricardo Torres ◽  
Vassiliy Bavro ◽  
Jessica Blair
Keyword(s):  
Western Blotting ◽  
Efflux Pump ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Phenotypic Effect ◽  
Resistance Nodulation Division ◽  
Binding Residues ◽  
Outer Membrane Channel

The resistance-nodulation-division (RND) family of efflux pumps confer clinically relevant antibiotic resistance in Gram-negative bacteria, such as Salmonella enterica. RND pumps, including AcrB, are organized as tri-partite systems, consisting of an inner membrane RND pump, a periplasmic adaptor protein (PAP) and an outer membrane channel. Previously, inactivation of the PAPs AcrA and AcrE in S. enterica has been shown to significantly increase susceptibility to antimicrobials and reduce virulence. Therefore, PAPs are seen as attractive targets for the development of efflux pump inhibitors. However, the role of PAPs in the assembly of tri-partite pumps and the residues involved in PAP-RND pump binding is poorly understood. In this study, point mutations in the predicted RND binding residues of AcrA were generated by site-directed mutagenesis. The point mutants were characterised phenotypically through ethidium bromide efflux assays and antimicrobial susceptibility testing. Furthermore, Western blotting was used to verify that the phenotypic effect of the point mutations was not due to destabilisation of the AcrA protein. Point mutations in certain residues, such as G58, F292, R294 and G363 were found to significantly impair efflux activity and increase susceptibility to various antibiotics and dyes, suggesting an important role for these AcrA residues in RND pump binding. Western blotting confirmed that these point mutants were stable and exhibited similar expression levels to the wild-type. These residues could be important targets for the design and development of PAP inhibitors to restore the activity of existing antibiotics and reduce virulence of Salmonella.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440

2020 ◽  
Vol 8 (11) ◽  
pp. 1782
Author(s):  
Tania Henriquez ◽  
Tom Baldow ◽  
Yat Kei Lo ◽  
Dina Weydert ◽  
Andreas Brachmann ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Efflux Pump ◽  
Toxic Metal ◽  
Growth Defect ◽  
Metal Chelators ◽  
Pseudomonas Putida Kt2440 ◽  
Pseudomonas Species ◽  
Resistance Nodulation Division ◽  
Rnd Efflux Pump

Bacteria must be able to cope with harsh environments to survive. In Gram-negative bacteria like Pseudomonas species, resistance-nodulation-division (RND) transporters contribute to this task by pumping toxic compounds out of cells. Previously, we found that the RND system TtgABC of Pseudomonas putida KT2440 confers resistance to toxic metal chelators of the bipyridyl group. Here, we report that the incubation of a ttgB mutant in medium containing 2,2’-bipyridyl generated revertant strains able to grow in the presence of this compound. This trait was related to alterations in the pp_2827 locus (homolog of mexS in Pseudomonas aeruginosa). The deletion and complementation of pp_2827 confirmed the importance of the locus for the revertant phenotype. Furthermore, alteration in the pp_2827 locus stimulated expression of the mexEF-oprN operon encoding an RND efflux pump. Deletion and complementation of mexF confirmed that the latter system can compensate the growth defect of the ttgB mutant in the presence of 2,2’-bipyridyl. To our knowledge, this is the first report on a role of pp_2827 (mexS) in the regulation of mexEF-oprN in P. putida KT2440. The results expand the information about the significance of MexEF-OprN in the stress response of P. putida KT2440 and the mechanisms for coping with bipyridyl toxicity.

scholarly journals Three Efflux Pumps Are Required To Provide Efficient Tolerance to Toluene in Pseudomonas putidaDOT-T1E

2001 ◽  
Vol 183 (13) ◽  
pp. 3967-3973 ◽  
Author(s):  
Antonia Rojas ◽  
Estrella Duque ◽  
Gilberto Mosqueda ◽  
Geir Golden ◽  
Ana Hurtado ◽  
...  
Keyword(s):  
Gas Phase ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Site Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Triple Mutant ◽  
Multidrug Efflux Pumps ◽  
Mutant Strains ◽  
Resistance Nodulation Division ◽  
Identification And Characterization

ABSTRACT In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgHmutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

scholarly journals Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

2004 ◽  
Vol 15 (8) ◽  
pp. 3591-3604 ◽  
Author(s):  
Espen Stang ◽  
Frøydis D. Blystad ◽  
Maja Kazazic ◽  
Vibeke Bertelsen ◽  
Tonje Brodahl ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Protein S ◽  
Egf Receptors ◽  
Ubiquitinated Protein ◽  
Coated Pits ◽  
Independent Manner ◽  
Sh3 Domains

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

scholarly journals Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

2018 ◽  
Author(s):  
Jiayun Liu ◽  
Wanliang Shi ◽  
Shuo Zhang ◽  
Gail Cassell ◽  
Dmitry A. Maslov ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Drug Targets ◽  
Drug Exposure ◽  
Efflux Pump ◽  
Active Form ◽  
Point Mutations ◽  
Drug Susceptibility ◽  
Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

scholarly journals Point mutations of two arginine residues in the Streptomyces R61 dd-peptidase

1992 ◽  
Vol 283 (1) ◽  
pp. 123-128 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Ghuysen ◽  
J M Frère
Keyword(s):  
Enzyme Activity ◽  
Enzyme Stability ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Arginine Residues

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

scholarly journals Exploring the role of RRM domains and conserved aromatic residues in RGG motif  of eIF4G-binding translation repressor protein Sbp1

2021 ◽  
Vol 3 ◽  
pp. 102
Author(s):  
Nupur Bhatter ◽  
Rajan Iyyappan ◽  
Gayatri Mohanan ◽  
Purusharth I Rajyaguru
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Interaction ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Growth Defect ◽  
Aromatic Residues ◽  
Over Expression ◽  
Rna Granules

Background: RNA binding proteins play crucial role in determining if a given mRNA will be translated, stored, or degraded. Sbp1 is an RGG-motif containing protein that is implicated in affecting mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In this report, we have assessed the genetic interaction of Sbp1 with decapping activators such as Dhh1, Pat1, and Scd6. We have further analyzed the importance of different domains and specific conserved residues of Sbp1 in its ability to cause over-expression mediated growth defect. Method: Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions were created in Sbp1 expressed under a galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon over-expression. The ability of Sbp1 to affect over-expression mediated growth defect of other decapping activators was tested using growth assay. Live cell imaging was done to study localization of Sbp1 and its RRM-deletion mutants to RNA granules upon glucose starvation. Results: Mutation of several aromatic residues in the RGG-motif and that of the phosphorylation sites in the RRM domain of Sbp1 did not affect the growth defect phenotype. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect the ability of Sbp1 to cause growth defect. Moreover, absence of Sbp1 did not affect the growth defect phenotypes observed upon overexpression of decapping activators Dhh1 and Pat1. Strikingly deletion of both the RRM domains (RRM1 and RRM2) and not the RNP motifs within them compromised the growth defect phenotype. Sbp1 mutant lacking both RRM1 and RRM2 was highly defective in localizing to RNA granules.   Conclusion: This study identifies an important role of RRM domains independent of the RNP motif in Sbp1 function.

scholarly journals Site-Directed Mutagenesis Reveals Putative Substrate Binding Residues in the Escherichia coli RND Efflux Pump AcrB

2008 ◽  
Vol 190 (24) ◽  
pp. 8225-8229 ◽  
Author(s):  
Jürgen A. Bohnert ◽  
Sabine Schuster ◽  
Markus A. Seeger ◽  
Eva Fähnrich ◽  
Klaas M. Pos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Point Mutation ◽  
Efflux Pump ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Rnd Efflux Pump ◽  
Binding Residues

ABSTRACT The Escherichia coli multidrug efflux pump protein AcrB has recently been cocrystallized with various substrates, suggesting that there is a phenylalanine-rich binding site around F178 and F615. We found that F610A was the point mutation that had the most significant impact on substrate MICs, while other targeted mutations, including conversion of phenylalanines 136, 178, 615, 617, and 628 to alanine, had smaller and more variable effects.

scholarly journals Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit.

1993 ◽  
Vol 123 (4) ◽  
pp. 1017-1025 ◽  
Author(s):  
L M Shaw ◽  
A M Mercurio
Keyword(s):  
Point Mutations ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Serine Phosphorylation ◽  
Laminin Receptor ◽  
Site Directed Mutagenesis ◽  
Receptor Function ◽  
Serine Residue ◽  
Beta 1 Integrin

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.

scholarly journals Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket

2008 ◽  
Vol 414 (3) ◽  
pp. 383-390 ◽  
Author(s):  
Mark Frazzetto ◽  
Cenk Suphioglu ◽  
Jiuxiang Zhu ◽  
Oleg Schmidt-Kittler ◽  
Ian G. Jennings ◽  
...  
Keyword(s):  
Point Mutations ◽  
Binding Pocket ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Specific Class ◽  
Pi3k Inhibitors ◽  
Isoform Selectivity ◽  
Phosphoinositide 3 Kinase ◽  
Conserved Amino Acids

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr886 and Lys890 of the PI3Kγ isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the β isoform (Glu858 and Asp862) and those in the α isoform (His855 and Gln859) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of β-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the α-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kβ activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the α-isoform-selectivity of the compounds studied.

Sign in / Sign up

Export Citation Format

Share Document