title: Structure and dynamics of Mycobacterium tuberculosis truncated hemoglobin N: insights from NMR spectroscopy and molecular dynamics simulations.
abstract: The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe²⁺-O₂ + (•)NO → trHbN-Fe³⁺-OH₂ + NO₃⁻) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (•)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (•)NO and O₂ to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.
authors: Département de biochimie, de microbiologie et de bio-informatique, Université Laval and PROTEO, Québec, Canada.
authors: Pierre-Yves
authors: PY
authors: Savard
authors: Richard
authors: R
authors: Daigle
authors: Sébastien
authors: S
authors: Morin
authors: Anne
authors: A
authors: Sebilo
authors: Fanny
authors: F
authors: Meindre
authors: Patrick
authors: P
authors: Lagüe
authors: Michel
authors: M
authors: Guertin
authors: Stéphane M
authors: SM
authors: Gagné
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D054793:Truncated Hemoglobins / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C102539:GlbN protein, bacteria; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D054793:Truncated Hemoglobins; D009569:Nitric Oxide; D014443:Tyrosine; D010105:Oxygenases; C114415:nitric oxide dioxygenase