Fig 1.
The distinctive truncated hemoglobin structure.
(A) Typical fold of a trHb structure and the commonly used structural blocks (shown in different colors). (B) Schematic representation showing the three different ligand entry tunnels present in trHbs: Long Tunnel (in red), Short Tunnel G8 (in blue) and E7 Gate (in green). (C) Schematic representation of the five structural positions that define the active site in a typical trHb. The figure shows the heme group (grey), the bound oxygen ligand (red in balls and sticks) and the five key residues (shown as sticks).
Fig 2.
Phylogenetic hallmarks of trHbs sequences.
(A) Bayesian phylogenetic tree of trHbs family presented as a radial phylogram. Dots indicate bayesian support of ≥80%. The scale bar represents a distance of 0.1 accepted amino acid substitutions per site. Red asterisks indicate sequences with resolved 3D structures that were used in the construction of both the multiple sequence alignment (MSA) used for phylogeny and the structural alignment presented in (B). (B) Structural alignment of 17 available trHbs sequences. The selected color regions, together with helix labeling, correspond with the most conserved regions in the MSA. (C) MI network of positions that have cMI > 65. Specificity determining position (SDP) E7 and its direct neighbors are highlighted. Color of the node is indicative for the Kullback-Leibler information. Red encircled nodes correspond with the central SDP, E7, and its direct neighbors. (D) Logo sequences trimmed with block mapping and gathering with entropy (BMGE) for all 1107 considered trHbs and for each separate group. One sequence member of each subgroup is also shown as a reference. Known hallmark and other important positions are shaded in grey with their structural position denominators indicated above, in red for active site residues and with yellow shading for identified SDPs.
Fig 3.
The three topological tunnels in trHbs.
Top: examples of free energy profiles (FEPs) along the three potential ligand migration paths for Mt-trHbN Long Tunnel (LT), Short Tunnel G8 (STG8) and Cj-trHbP at E7 Gate (E7G). DP, SP and TP correspond to distal, secondary and tertiary pockets, respectively. The x coordinate is defined by the Fe-O2 distance through the pathway. Bottom: schematic representations of the heme distal residues, the tunnel and cavities system estimated with implicit ligand sampling for LT, STG8 and E7G.
Fig 4.
Plot of log(konexp) vs log(koncalc).
Dots indicate Mb in red and the trHbs in green, blue and orange for groups N, O and P, respectively. Dots with lighter colors indicate mutant proteins. Identity line is shown in grey. A complete list of experimental and calculated values is available at S3 Table.
Fig 5.
Plot of log(koffexp) vs log(koffcalc).
Dots indicate Mb in red and the trHbs in green, blue and orange for groups N, O and P, respectively. Other globin forms are indicated in purple. Circles with lighter colors indicate mutant proteins. Identity line is shown in grey. A complete list of experimental and calculated values is available at S4 Table.
Fig 6.
Collapsed phylogenetic tree of trHbs annotated with their key physicochemical characteristics.
The circular phylogram shows the topology derived from Fig 2A, with color-matched boxes indicating hierarchical clustering. values are mapped on the inner concentric circle using a violet color gradient (greater stabilization, darker violet). Tunnel apertures are mapped as red, cyan and light green dots for Long Tunnel, Short Tunnel G8 and E7 Gate, respectively, on the other concentric circles. Dot size indicates tunnel openness.