The N-terminal pre-A region of Mycobacterium tuberculosis 2/2HbN promotes NO-dioxygenase activity.
A unique defense mechanisms by which Mycobacterium tuberculosis protects itself from nitrosative stress is based on the O2 -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-ΔpreA, lacking the pre-A region, at a resolution of 1.53 Å. 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-ΔpreA 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 O2 -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 kon value for peroxynitrite isomerization by Mt2/2HbN-ΔpreA-Fe(III) is four-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-ΔpreA-Fe(II)-O2 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.


METHODS
Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 5AB8.
Department of Physics, University of Genova, Italy.
Alessandra
A
Pesce
Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Argentina.
Juan P
JP
Bustamante
Departament de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, University of Barcelona, Santa Coloma de Gramenet, Spain.
Axel
A
Bidon-Chanal
Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Argentina.
Leonardo
L
Boechi
Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Argentina.
Darío A
DA
Estrin
Departament de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, University of Barcelona, Santa Coloma de Gramenet, Spain.
Francisco Javier
FJ
Luque
Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Quebec, Canada.
Anne
A
Sebilo
Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Quebec, Canada.
Michel
M
Guertin
Department of Biosciences, University of Milan, Italy.|CNR-IBF and CIMAINA, University of Milan, Italy.
Martino
M
Bolognesi
Interdepartmental Laboratory of Electron Microscopy, Roma Tre University, Rome, Italy.|National Institute of Biostructures and Biosystems, Rome, Italy.
Paolo
P
Ascenzi
Department of Biosciences, University of Milan, Italy.
Marco
M
Nardini
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D054793:Truncated Hemoglobins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D054793:Truncated Hemoglobins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D006418:Heme; D049308:Dioxygenases
2/2 hemoglobins; NO dioxygenase; globin dynamics; heme/ligand tunneling; truncated hemoglobins