Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

Boron, Ignacio; Bustamante, Juan Pablo; Davidge, Sandra T.; Singh, Sakshi; Bowman, Lesley A.H.; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radí, Rafael; Poole, Robert K.; Dikshit, Kanak L.; Estrin, Darı́o A.; Marti, Marcelo Adrian; Boechi, Leonardo

doi:10.12688/f1000research.5921.2

Cited by 13 publications

(10 citation statements)

References 39 publications

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“…Moreover, the double mutant, where there are no more water stabilizing interactions, results in an even larger increase in k on . To show the role played by the tunnel, we can look at the ValG8Phe mutant of Mt-trHbN, which has a 10 times smaller k on compared to the wt [ 36 ] having very similar distal site residues, and thus similar . However, the ligand needs to overcome higher barriers along the tunnel to reach the active site, mainly due to the size increase of G8 residue (see Figs 4A and 3B ), a fact that is reflected in smaller k tunnels .…”

Section: Resultsmentioning

confidence: 99%

“…Free energy profiles (FEP) for the O 2 migration process along the protein tunnel/cavity system were computed using the Implicit Ligand Sampling (ILS) approach [ 77 ], which has been widely used to study these process and was shown previously by our group to yield accurate results [ 20 , 36 , 78 ]. ILS calculations were performed in a rectangular grid (0.5 Å resolution) that includes the whole simulation box (i.e.…”

Section: Methodsmentioning

confidence: 99%

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Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

et al. 2016

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Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

et al. 2016

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“…In support, a TrpE15 variant showed two- to threefold less NOD activity when reductively coupled and assayed within E. coli ( 30 ) but inexplicably showed no effect on NO dioxygenation by HbNFe 3+ O 2 − ( 32 ) and little apparent effect on O 2 or NO migration to the ferrous heme ( 30 ). On the other hand, replacing LeuG8 with Phe or Trp caused large effects on CO rebinding kinetics ( 33 ), suggesting a preferential pathway for CO migration. Molecular dynamics simulations of HbN map a probable escape route for NO 3 − at the ThrE2 residue in the CD loop ( 34 ).…”

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Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin

Gardner

2021

Journal of Biological Chemistry

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The substrates O 2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O 2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O 2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains trigger the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH 2 to reduce the ferric iron, which forms the stable ferric–superoxide–TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 10 10 M −1 s −1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O 2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.

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“…The diameter of the tunnel bottleneck is estimated to be approximately 2.5 Å, suggesting that the thermal fluctuations should a priori enable the passage of gaseous ligands. Noteworthy, Val(94)G8 has been shown to play a key role in the control of ligand association in truncated Hbs .…”

Section: Resultsmentioning

confidence: 99%

“…In particular Phe(62)E15, a tunnel long branch residue, can adopt two conformations where its phenyl side chain may block the tunnel branch (the so‐called closed state) or not (the open state) . However, the results of a recent study suggest that the so‐called closed state does not fully prevent transit of ligands .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

show abstract

Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

Cited by 13 publications

References 39 publications

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin

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

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