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

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-DpreA, lacking the pre-A region, at a resolution of 1.53A. 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-DpreA 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-DpreA-Fe(III) is four-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-DpreA-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.Abbreviations 2/2Hb, 2-on-2 globin; At2/2AHb3, 2/2AHb3 from Arabidopsis thaliana; DLS, dynamic light scattering; FdR, NADH-flavodoxin reductase; ILS, implicit ligand sampling; MD, molecular dynamics; Mt2/2Hb, Mycobacterium tuberculosis 2/2Hb; Mt2/2HbN-DpreA, Mt2/2HbN deletion mutant devoid of the first 11 amino acids; NOD, nitric oxide dioxygenase; PDB, Protein Data Bank.

Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

“…The main features of Ph-2/2HbO-2217 and Ph-2/2HbO-0030 are the presence of a longer sequence extension of the N-terminal region (19 residues in Ph-2/2HbO-0030 and 9 residues in Ph-2/2HbO-2217), that in Ph-2/2HbO-0030 is proteolytically cleaved during protein purification (Giordano et al, 2007), and does not appear to reduce the NO scavenging activity (Coppola et al, 2013). Pesce et al (2016) have recently demonstrated that removal of the pre-A region in M.…”

“…Since the 2/2Hbs scaffold probably evolved before the development of the current aerobic environment, a putative original role for these proteins could have been that of O2 detoxification following the increase of O2 levels and the evolution of photosynthesis (Crowe et al, 2013). The search for globin physiological functions is further driven by the evidence that many 2/2Hbs are capable of reacting with NO, nitrite, and peroxynitrite (Gardner, 2005;Ascenzi et al, 2009;De Marinis et al, 2009;Ascenzi et al, 2014;Pesce et al, 2016). Interestingly, an unusual occurrence of the concomitant presence of 2/2Hbs of group II and flavohemoglobin (FHb) in the same genome has been demonstrated (Vinogradov et al, 2013).…”

Distinctive structural properties of THB11, a pentacoordinate Chlamydomonas reinhardtii truncated hemoglobin with N- and C-terminal extensions

“…Although our current data do not allow us to distinguish between a role of the C-terminus by itself or by its influence on the oligomerization status, extensions of the globin domain have been shown before to influence the reactivities of globins, such as the pre-A motif of M. tuberculosis GlbN, which is crucial for the high NO dioxygenase activity of the protein. It has been proposed that its absence influences protein dynamics in a way that the PheE15 gate constitutively blocks the long tunnel [88], whereas more recent results suggest that the pre-A motif prevents the formation of stable GlbN dimers, in which the short tunnel is blocked, and the long tunnel restricted [89]. In Methanosarcina acetivorans protoglobin, a 3/3Hb, dimerization has instead been calculated to result in an opening of the tunnels [90].…”

Multidomain truncated hemoglobins: New members of the globin family exhibiting tandem repeats of globin units and domain fusion

“…However, these trHb1_N domains lacked the Nterminal pre-A region ( Fig. 2A) that is present in the trHb1_N of M. tuberculosis and modulates its NO-dioxygenase activity (38)(39)(40)(41), thereby suggesting the role of multidomain trHb1_N in other cellular functions.…”