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Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

Boron I, Bustamante JP, Davidge KS, Singh S, Bowman LA, Tinajero-Trejo M, Carballal S, Radi R, Poole RK, Dikshit K, Estrin DA, Marti MA, Boechi L - F1000Res (2015)

Bottom Line: In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W.These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site.We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, C1428EGA, Argentina.

ABSTRACT
Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

No MeSH data available.


Related in: MedlinePlus

Apparent rateskobs for CO binding to ferrous Mt-trHbN.Curves for wild type (green), VG8F (orange) and VG8W (violet) mutants as a function of CO concentration in stopped flow measurements are shown. The time courses are measured at different CO concentrations ranging from 10 to 200 µM (after mixing). Continuous line corresponds to linear fit ofkobs rates.
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f3: Apparent rateskobs for CO binding to ferrous Mt-trHbN.Curves for wild type (green), VG8F (orange) and VG8W (violet) mutants as a function of CO concentration in stopped flow measurements are shown. The time courses are measured at different CO concentrations ranging from 10 to 200 µM (after mixing). Continuous line corresponds to linear fit ofkobs rates.

Mentions: Although CO is not the natural ligand of the hemeproteins, it is widely used as a probe for ligand association studies due to its ease of use. In order to address the molecular determinants controlling ligand migration we performed CO ligand association constant measurements of wild type Mt-trHbN and two mutants: VG8F and VG8W. Kinetic traces for CO binding were measured through the absorption changes at the CO adduct peak position (λ=423 nm;Figure 2). Association of CO is well described by a single exponential decay, whose rate constant (kobs) depends linearly on CO concentration and the slope can be interpreted askon CO. A significantkon CO decrease for VG8F (715 ± 27 mM-1s-1), and an even larger decrease for VG8W (48 ± 1 mM-1s-1) was observed in relation to that observed for the wild type protein (4495± 357 mM-1s-1) (Figure 3).Table 1 summarizes the measuredkon CO values for wild type and mutant Mt-trHbs O and N, and is presented alongside literature data.


Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

Boron I, Bustamante JP, Davidge KS, Singh S, Bowman LA, Tinajero-Trejo M, Carballal S, Radi R, Poole RK, Dikshit K, Estrin DA, Marti MA, Boechi L - F1000Res (2015)

Apparent rateskobs for CO binding to ferrous Mt-trHbN.Curves for wild type (green), VG8F (orange) and VG8W (violet) mutants as a function of CO concentration in stopped flow measurements are shown. The time courses are measured at different CO concentrations ranging from 10 to 200 µM (after mixing). Continuous line corresponds to linear fit ofkobs rates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591903&req=5

f3: Apparent rateskobs for CO binding to ferrous Mt-trHbN.Curves for wild type (green), VG8F (orange) and VG8W (violet) mutants as a function of CO concentration in stopped flow measurements are shown. The time courses are measured at different CO concentrations ranging from 10 to 200 µM (after mixing). Continuous line corresponds to linear fit ofkobs rates.
Mentions: Although CO is not the natural ligand of the hemeproteins, it is widely used as a probe for ligand association studies due to its ease of use. In order to address the molecular determinants controlling ligand migration we performed CO ligand association constant measurements of wild type Mt-trHbN and two mutants: VG8F and VG8W. Kinetic traces for CO binding were measured through the absorption changes at the CO adduct peak position (λ=423 nm;Figure 2). Association of CO is well described by a single exponential decay, whose rate constant (kobs) depends linearly on CO concentration and the slope can be interpreted askon CO. A significantkon CO decrease for VG8F (715 ± 27 mM-1s-1), and an even larger decrease for VG8W (48 ± 1 mM-1s-1) was observed in relation to that observed for the wild type protein (4495± 357 mM-1s-1) (Figure 3).Table 1 summarizes the measuredkon CO values for wild type and mutant Mt-trHbs O and N, and is presented alongside literature data.

Bottom Line: In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W.These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site.We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, C1428EGA, Argentina.

ABSTRACT
Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

No MeSH data available.


Related in: MedlinePlus