Ligand Interactions in the Distal Heme Pocket of <i>Mycobacterium tuberculosis</i> Truncated Hemoglobin N: Roles of TyrB10 and GlnE11 Residues^†

The crystallographic structure of oxygenated trHbN from Mycobacterium tuberculosis showed an extended heme distal site hydrogen-bonding network that includes Y(B10), Q(E11), and the bound O2 (Milani, M., et al. (2001) EMBO J. 20, 3902−3909). In the present work, we analyze the effects that substitutions at the B10 and E11 positions exert on the heme and its coordinated ligands, using steady-state resonance Raman spectroscopy, absorption spectroscopy and X-ray crystallography. Our results show that (1) residues Y(B10) and Q(E11) control the binding and the ionization state of the heme-bound water molecules in ferric trHbN and are important in keeping the sixth coordination position vacant in deoxy trHbN; (2) residue Q(E11) plays a role in maintaining the integrity of the proximal Fe−His bond in deoxy trHbN; (3) in wild-type oxy-trHbN, the size and hydrogen-bonding capability of residue E11 is important to sustain proper interaction between Y(B10) and the heme-bound O2; (4) CO-trHbN is in a conformational equilibrium, where either the Y(B10) or the Q(E11) residue interacts with the heme-bound CO; and (5) Y(B10) and Q(E11) residues control the conformation (and likely the dynamics) of the protein matrix tunnel gating residue F(E15). These findings suggest that the functional processes of ligand binding and diffusion are controlled in trHbN through the dynamic interaction of residues Y(B10), Q(E11), F(E15), and the heme ligand

Data collection and refinement statistics.

<p>Values in parentheses refer to the highest resolution shells.</p><p>*Rwork = Σhkl||Fo|—|Fc|| ⁄ Σhkl|Fo| for all data, except 10%, which were used for Rfree calculation.</p><p>^#Average temperature factors over the whole structure.</p><p>Data collection and refinement statistics.</p

Thermodynamic stability of β2m D-to-N variants.

<p>(A) Comparison of the CD spectra of wt β2m and D-to-N variants. (B) Thermal stability assessed by CD experiments in the far-UV region. (C) Tm values are determined as the minima of the first derivative of the unfolding profiles, each thermal unfolding was repeated four times. Standard deviations were calculated and are shown for each Tm. Arrows highlight graphically the Tm differences.</p

Aggregation of wt β2m and of D-to-N variants.

<p>Under shaking conditions in physiological buffer, an increase of ThT signal was recorded solely for D76N β2m (red), no signal increase was registered for the wt protein (black) or the other variants, whose signals are comprised in the base line: D34N (brown), D38N (orange), D53N (blue), D59N (grey), D96N (pink), D98N (green).</p

Comparison of the crystal structures of D-to-N β2m variants.

<p>(A), (C) and (E) Cartoon representation of the β2m variants D53N (yellow), D59N (blue) and D98N (cyan) individually overlaid onto the structure of wt β2m (light grey, PDB ID 2YXF). (B), (D) and (F) Close up views of the mutation sites in D53N, D59N and D98N, color coded as in A C and E panels; side chains of the mutated and neighboring residues are shown as sticks and labeled.</p

Comparison of the B_z-score profiles for wt β2m and four D-to-N mutants.

<p>Plot of the B_z-score<i>versus</i> the residue number. Data for wt β2m (red, pdb ID 2YXF), D76N (black, pdb ID 4FXL), D38N (cyan), D53N (green), D59N (purple), and D98N (orange) are presented. β-strands building up the β2m fold are shown as arrows, and labelled in the lower part of the graph.</p

Distribution and conservation of Asp residues in β2m.

<p>(A) Asp residues are mapped on a ribbon representation of wt β2m structure (pdb code 2YXF), and shown as stick models. (B) Alignment of mammalian β2m amino acid sequences, numbering according to the mature human protein; D76 is highlighted in green, while other conserved Asp residues are highlighted in yellow. Asp residues randomly located in different sequences are shown in cyan.</p

Values of the second-order rate constant for peroxynitrite isomerization by ferric heme-proteins.

a<p>pH 7.4 and 20°C. Present study.</p>b<p>pH 7.0 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi7" target="_blank">[32]</a>.</p>c<p>pH 7.0 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Coppola1" target="_blank">[31]</a>.</p>d<p>pH 7.0 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Herold2" target="_blank">[22]</a>.</p>e<p>pH 7.5 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Herold4" target="_blank">[24]</a>.</p>f<p>pH 7.5 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Herold2" target="_blank">[22]</a>.</p>g<p>pH 7.2 and 22°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi4" target="_blank">[28]</a>.</p>h<p>pH 7.0 and 20°C. Cardiolipin was 1.6×10^–4 M. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi5" target="_blank">[29]</a>.</p

RMSD values (Å) for the 3D structural comparisons of the D-to-N mutants with wt and D76N β2m (pdb code 2YXF and 4FXL, respectively).

<p>* Atoms belonging to AB loops were excluded in this calculation</p><p>The number of Cα atoms used for each calculation is reported in parenthesis.</p

Ivabradine block of WT and F509A channels in the open state.

<p>A, representative traces showing the action of ivabradine (3 and 30 µM) on the current recorded from WT and F509A channels during a long (100 s) step to −100 mV. B, dose-response relations for ivabradine block measured as in A. Hill fitting resulted in IC₅₀, nH values of 120.7 µM, 0.5 and 91.3 µM, 0.5 for WT (filled circles) and F509A channels (open circles), respectively (non-significantly different, P>0.05).</p