Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

Abstract

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O2/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Understanding the kinetics of ligand binding to globins with molecular dynamics simulations: the necessity of multiple state models

“…Some of them also present gating residues, like the nonsymbiotic haemoglobin AHb1 from Arabidopsis thaliana, or the truncated haemoglobin N (trHbN) from Mycobacterium tuberculosis, while others present tunnel systems free of any blocking residues, like the Cerebratulus lacteus minihaemoglobin (mini-Hb) ( Fig. 1) [17][18][19][20]. The presence of small pockets where a small gaseous ligand can migrate once it is photolysed from the haem has been reported also for other globins like neuroglobin or cytoglobin (Cygb), as well as that thermal fluctuations modulate the accessibility of these cavities [21,22].…”

Mechanistic Insight into the Enzymatic Reduction of Truncated Hemoglobin N of Mycobacterium tuberculosis

“…Briefly, the flavin cofactor of the protein was extracted after boiling for 5 min and centrifugation at 14,000 ϫ g. FAD content was determined through fluorescence at 520 nm with excitation at 460 nm using an extinction coefficient of 11.6 mM Ϫ1 cm Ϫ1 . Titration of oxygenated MtbHbN and its mutants by NO was checked essentially as described earlier (8,12), and profiles of NO-induced oxidation were recorded after each addition of NO for the conversion of oxygenated HbN into the oxidized state.…”

“…MtbHbN backbone to carry out the NOD function, whereas its deletion compromises the NOD function due to the hindered backbone motion (8,12). The main difference in the dynamics of protein structure was observed around B and E helices and the movement of CD and EF loop regions, suggesting that Pre-A deletion may affect the intermolecular interactions of HbN with FdR and the efficiency of HbN reduction.…”

“…In particular, previous studies showed that the Pre-A-deleted HbN displayed a clear cut restriction in the movement of B and E helices as well as the EF loop region (16), which in conjunction with the CD loop provides key residues involved in the recognition of HbN and FdR. It can, therefore, be concluded that the Pre-A segment influences the NOD function by modulating the dynamics of the protein core, which in turn influences two distinct effects: (i) the relative motion between helices B and E favors the conformational changes of the Phe(E15) gate, thus favoring the transition between closed and open states (8,12), and (ii) the enhanced flexibility of helices B and E regulates movement of the CD loop and facilitates the interactions between residues that modulate the appropriate recognition at the protein-protein interface. Thus, the results derived from MD simulations reveal the formation of salt bridges supported by residues located in helix B (Glu 25 , Glu 29 ) and E (Arg The HbN may have significant implications on the pathophysiology of Mtb due to its high oxygen affinity and NO detoxification ability, which may be crucial for sustaining its intracellular survival within the NO-enriched hypoxic environment of macrophages and granuloma.…”

“…Raman spectroscopy revealed that heme iron coordination in MtbHbN is well suited for performing O 2 /NO chemistry for NO dioxygenation (7), which is modulated by two unusual structural features of the HbN: a 12-residue long highly flexible N-terminal Pre-A region that is required for the optimal NOD activity (8), and a protein tunnel system composed of short and long branches (9) that facilitates ligand entry to the distal heme site (9,10). The functional role of the tunnel has been underlined in a dual path mechanism for ligand access to the heme cavity (11), in which binding of O 2 to the heme triggers dynamical alterations in the protein backbone that regulate the opening of the long branch via a phenylalanine gate (Phe(E15)) (11,12), thus facilitating access of NO to the O 2 -bound heme.…”