A unique defense mechanisms by which Mycobacterium tuberculosis protects itself from nitrosative stress is based on the O-2-dependent NO-dioxygenase (NOD) activity of truncated hemoglobin 2/2HbN (Mt2/2HbN). The NOD activity largely depends on the efficiency of ligand migration to the heme cavity through a two-tunnel (long and short) system; recently, it was also correlated with the presence at the Mt2/2HbN N-terminus of a short pre-A region, not conserved in most 2/2HbNs, whose deletion results in a drastic reduction of NO scavenging. In the present study, we report the crystal structure of Mt2/2HbN-Delta preA, lacking the pre-A region, at a resolution of 1.53 angstrom. We show that removal of the pre-A region results in long range effects on the protein C-terminus, promoting the assembly of a stable dimer, both in the crystals and in solution. In the Mt2/2HbN-Delta preA dimer, access of heme ligands to the short tunnel is hindered. Molecular dynamics simulations show that the long tunnel branch is the only accessible pathway for O-2-ligand migration to/from the heme, and that the gating residue Phe(62) E15 partly restricts the diameter of the tunnel. Accordingly, kinetic measurements indicate that the k(on) value for peroxynitrite isomerization by Mt2/2HbN-Delta preA-Fe(III) is four-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-Delta preA-Fe(II)-O-2 is reduced by 35-fold. Therefore, we speculate that Mt2/2HbN evolved to host the pre-A region as a mechanism for preventing dimerization, thus reinforcing the survival of the microorganism against the reactive nitrosative stress in macrophages.

Pesce, A., Bustamante, J.p., Bidon-Chanal, A., Boechi, L., Estrin, D.a., Luque, F.j., et al. (2016). The N-terminal pre-A region of Mycobacterium tuberculosis 2/2HbN promotes NO-dioxygenase activity. FEBS JOURNAL. S, 283(2), 305-322 [10.1111/febs.13571].

The N-terminal pre-A region of Mycobacterium tuberculosis 2/2HbN promotes NO-dioxygenase activity

ASCENZI, Paolo;
2016

Abstract

A unique defense mechanisms by which Mycobacterium tuberculosis protects itself from nitrosative stress is based on the O-2-dependent NO-dioxygenase (NOD) activity of truncated hemoglobin 2/2HbN (Mt2/2HbN). The NOD activity largely depends on the efficiency of ligand migration to the heme cavity through a two-tunnel (long and short) system; recently, it was also correlated with the presence at the Mt2/2HbN N-terminus of a short pre-A region, not conserved in most 2/2HbNs, whose deletion results in a drastic reduction of NO scavenging. In the present study, we report the crystal structure of Mt2/2HbN-Delta preA, lacking the pre-A region, at a resolution of 1.53 angstrom. We show that removal of the pre-A region results in long range effects on the protein C-terminus, promoting the assembly of a stable dimer, both in the crystals and in solution. In the Mt2/2HbN-Delta preA dimer, access of heme ligands to the short tunnel is hindered. Molecular dynamics simulations show that the long tunnel branch is the only accessible pathway for O-2-ligand migration to/from the heme, and that the gating residue Phe(62) E15 partly restricts the diameter of the tunnel. Accordingly, kinetic measurements indicate that the k(on) value for peroxynitrite isomerization by Mt2/2HbN-Delta preA-Fe(III) is four-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-Delta preA-Fe(II)-O-2 is reduced by 35-fold. Therefore, we speculate that Mt2/2HbN evolved to host the pre-A region as a mechanism for preventing dimerization, thus reinforcing the survival of the microorganism against the reactive nitrosative stress in macrophages.
Pesce, A., Bustamante, J.p., Bidon-Chanal, A., Boechi, L., Estrin, D.a., Luque, F.j., et al. (2016). The N-terminal pre-A region of Mycobacterium tuberculosis 2/2HbN promotes NO-dioxygenase activity. FEBS JOURNAL. S, 283(2), 305-322 [10.1111/febs.13571].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11590/304450
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