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. 2016 Jan 20;12(1):e1004701.
doi: 10.1371/journal.pcbi.1004701. eCollection 2016 Jan.

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

Juan P Bustamante  1 , Leandro Radusky  2 , Leonardo Boechi  3 , Darío A Estrin  1 , Arjen Ten Have  4 , Marcelo A Martí  3
Affiliations

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

Juan P Bustamante et al. PLoS Comput Biol. .

Abstract

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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
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
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
Fig 4. Plot of log(k on exp) vs log(k on calc).
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
Fig 5. Plot of log(k off exp) vs log(k off calc).
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
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. ΔΔEO2 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.
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