The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Edward T Lally - One of the best experts on this subject based on the ideXlab platform.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Abstract Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1, LFA-1) results in the clustering of the toxin/receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by four orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, while CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins. Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid Consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis.
Angela C Brown - One of the best experts on this subject based on the ideXlab platform.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Abstract Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1, LFA-1) results in the clustering of the toxin/receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by four orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, while CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins. Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid Consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis.
Diane E Frank - One of the best experts on this subject based on the ideXlab platform.
-
thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator effects of converting a Consensus Site to a non specific Site
Journal of Molecular Biology, 1997Co-Authors: Diane E Frank, Ruth M Saecker, Jeffrey P Bond, Michael W Capp, Oleg V Tsodikov, Sonya E Melcher, Mark M Levandoski, Thomas M RecordAbstract:Abstract What are the thermodynamic consequences of the stepwise conversion of a highly specific (Consensus) protein-DNA interface to one that is non-specific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific Site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (Consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA Sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 Download : Download full-size image in each 10 bp half Site of the symmetric lac operator (Osym). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the Consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (Kobs) for Osym by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the Consensus Site, the free energy effects of multiple substitutions are non-additive: the first reduces |ΔGobso| by 3 to 5 kcal mol−1, approximately halfway to the non-specific level, whereas the second is less deleterious, reducing |ΔGobso| by less than 3 kcal mol−1. Variant-specific dependences of Kobs on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of Osym and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, ΔCobso, is large and negative. In general, |ΔCobso| decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with Osym and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for Osym, but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (ΔΔGobso). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a Consensus interface to a less specific one; such insights are not obtained from comparisons at the level of ΔΔGobso. We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA Site) which accompany binding.
-
thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator effects of converting a Consensus Site to a non specific Site
Journal of Molecular Biology, 1997Co-Authors: Diane E Frank, Ruth M Saecker, Jeffrey P Bond, Michael W Capp, Oleg V Tsodikov, Sonya E Melcher, Mark M Levandoski, M T RecordAbstract:What are the thermodynamic consequences of the stepwise conversion of a highly specific (Consensus) protein-DNA interface to one that is nonspecific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific Site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (Consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA Sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 [sequence: see text] in each 10 bp half Site of the symmetric lac operator (Osym). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the Consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (K(obs)) for Osym by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the Consensus Site, the free energy effects of multiple substitutions are non-additive: the first reduces /deltaG(obs)o/ by 3 to 5 kcal mol(-1), approximately halfway to the non-specific level, whereas the second is less deleterious, reducing /deltaG(obs)o/ by less than 3 kcal mol(-1). Variant-specific dependences of K(obs) on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of Osym and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, deltaC(obs)o, is large and negative. In general, /deltaC(obs)o/ decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with Osym and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for Osym, but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (delta deltaG(obs)o). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a Consensus interface to a less specific one; such insights are not obtained from comparisons at the level of delta deltaG(obs)o. We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA Site) which accompany binding.
Nataliya V Balashova - One of the best experts on this subject based on the ideXlab platform.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Abstract Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1, LFA-1) results in the clustering of the toxin/receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by four orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, while CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins. Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid Consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis.
Richard M Epand - One of the best experts on this subject based on the ideXlab platform.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Abstract Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1, LFA-1) results in the clustering of the toxin/receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by four orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, while CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
-
aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition amino acid Consensus Site for membrane association
Journal of Biological Chemistry, 2013Co-Authors: Angela C Brown, Nataliya V Balashova, Richard M Epand, Raquel F Epand, Alvina Bragin, Scott C Kachlany, Michael J Walters, Kathleen Boeszebattaglia, Edward T LallyAbstract:Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid Consensus (CRAC) Sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 Site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins. Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid Consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis.