Ion pathway. Alternatively, receptor conformations that are stabilized by Lys in position 82 may have decreased affinity for HIV Env or decreased get KDM5A-IN-1 ability to induce the fusogenic Env protein conformation that mediates membrane fusion. In terms of the ensemble model of receptor conformation [3,62], mutation of Thr2.56(82) to Lys, may stabilize an ensemble of CCR5 conformations that Tartrazine web includes the micro-conformations that activate G proteins and receptor internalization, but not the micro-conformations that induce Env-directed membrane fusion. In contrast, mutation of Thr2.56(82) to Pro appears to stabilize an ensemble of receptor conformations that activate G protein and mediate the co-receptor functions of CCR5, but do not activate internalization (Fig. 4). Distinct activated conformations of CCR5 with differential abilities to support HIV Env-directed membrane fusion opens the possibility of developing CCR5 ligands that select specific receptor conformations. Indeed, a 25331948 recent comparison of the CCR5 blockers, TAK 779 and maraviroc, has shown that maraviroc has higher antiviral potency that does not correlate with inverseFigure. 4. Venn diagram depicting ensembles of CCR5 receptor conformations stabilized by mutation of Thr2.56(82). Triangles represent receptor conformations stabilized by mutation of Thr2.56(82) to Lys or Pro. Circles represent receptor conformations that mediate G protein activation, receptor internalization or HIV Env-directed membrane fusion. Mutation of Thr2.56(82) to Lys stabilizes an ensemble of receptor conformations that activate G protein-mediated signaling and conformations with increased susceptibility to internalization, but not conformations that support HIV Env dependent membrane fusion. The Thr2.56(82)Pro mutation stabilizes an ensemble of receptor conformations that activate the G protein and conformations that support HIV-1 fusion, but it does not appear to increase population of receptor conformations that result in decreased membrane expression of CCR5. doi:10.1371/journal.pone.0054532.gagonist activity or ability to block gp120 binding. It was suggested that maraviroc may selectively destabilize CCR5 conformations that trigger Env penetration of cell membranes [63]. Furthermore, it has been shown that CCR5 heterodimerizes with the CXCR4 co-receptor and that antagonists specific for one receptor allosterically cross-inhibit ligand binding and agonist function at the other receptor [64]. This raises the potential that CCR5blocking drugs may be developed to cross-inhibit infection by X4tropic viruses in cells where both receptors are expressed. In conclusion, we have shown that charge-neutralizing mutations of the Asp3.49(125) residue of the DRY motif do not result in 23977191 constitutive activity of CCR5, confirming that the CCR5 receptor does not conform to the consensus mechanism of receptor activation. We have confirmed that Lys or Pro substitutions for the Thr2.56(82) residue of the TxP motif cause constitutive activity of CCR5, but we have shown that mutants have distinct properties. Constitutively active mutants with Lys in position 82 show decreased cell surface expression and decreased HIV coreceptor function, whereas mutants with Pro in position 82 were well expressed and fully functional as HIV co-receptors. These distinct properties suggest that the mutations stabilize ensembles of receptor conformations that differ in their ability to induce the fusogenic HIV Env conformation. Our results suggest that.Ion pathway. Alternatively, receptor conformations that are stabilized by Lys in position 82 may have decreased affinity for HIV Env or decreased ability to induce the fusogenic Env protein conformation that mediates membrane fusion. In terms of the ensemble model of receptor conformation [3,62], mutation of Thr2.56(82) to Lys, may stabilize an ensemble of CCR5 conformations that includes the micro-conformations that activate G proteins and receptor internalization, but not the micro-conformations that induce Env-directed membrane fusion. In contrast, mutation of Thr2.56(82) to Pro appears to stabilize an ensemble of receptor conformations that activate G protein and mediate the co-receptor functions of CCR5, but do not activate internalization (Fig. 4). Distinct activated conformations of CCR5 with differential abilities to support HIV Env-directed membrane fusion opens the possibility of developing CCR5 ligands that select specific receptor conformations. Indeed, a 25331948 recent comparison of the CCR5 blockers, TAK 779 and maraviroc, has shown that maraviroc has higher antiviral potency that does not correlate with inverseFigure. 4. Venn diagram depicting ensembles of CCR5 receptor conformations stabilized by mutation of Thr2.56(82). Triangles represent receptor conformations stabilized by mutation of Thr2.56(82) to Lys or Pro. Circles represent receptor conformations that mediate G protein activation, receptor internalization or HIV Env-directed membrane fusion. Mutation of Thr2.56(82) to Lys stabilizes an ensemble of receptor conformations that activate G protein-mediated signaling and conformations with increased susceptibility to internalization, but not conformations that support HIV Env dependent membrane fusion. The Thr2.56(82)Pro mutation stabilizes an ensemble of receptor conformations that activate the G protein and conformations that support HIV-1 fusion, but it does not appear to increase population of receptor conformations that result in decreased membrane expression of CCR5. doi:10.1371/journal.pone.0054532.gagonist activity or ability to block gp120 binding. It was suggested that maraviroc may selectively destabilize CCR5 conformations that trigger Env penetration of cell membranes [63]. Furthermore, it has been shown that CCR5 heterodimerizes with the CXCR4 co-receptor and that antagonists specific for one receptor allosterically cross-inhibit ligand binding and agonist function at the other receptor [64]. This raises the potential that CCR5blocking drugs may be developed to cross-inhibit infection by X4tropic viruses in cells where both receptors are expressed. In conclusion, we have shown that charge-neutralizing mutations of the Asp3.49(125) residue of the DRY motif do not result in 23977191 constitutive activity of CCR5, confirming that the CCR5 receptor does not conform to the consensus mechanism of receptor activation. We have confirmed that Lys or Pro substitutions for the Thr2.56(82) residue of the TxP motif cause constitutive activity of CCR5, but we have shown that mutants have distinct properties. Constitutively active mutants with Lys in position 82 show decreased cell surface expression and decreased HIV coreceptor function, whereas mutants with Pro in position 82 were well expressed and fully functional as HIV co-receptors. These distinct properties suggest that the mutations stabilize ensembles of receptor conformations that differ in their ability to induce the fusogenic HIV Env conformation. Our results suggest that.
