Atomic-Level View of the Functional Transition in Vertebrate Hemoglobins: The Case of Antarctic Fish Hbs

Abstract

Tetrameric hemoglobins (Hbs) are prototypal systems for studies aimed at unveiling basic structure-function relationships as well as investigating the molecular/structural basis of adaptation of living organisms to extreme conditions. However, a chronological analysis of decade-long studies conducted on Hbs is illuminating on the difficulties associated with the attempts of gaining functional insights from static structures. Here, we applied molecular dynamics (MD) simulations to explore the functional transition from the T to the R state of the hemoglobin of the Antarctic fish Trematomus bernacchii (HbTb). Our study clearly demonstrates the ability of the MD technique to accurately describe the transition of HbTb from the T to R-like states, as shown by a number of global and local structural indicators. A comparative analysis of the structural states that HbTb assumes in the simulations with those detected in previous MD analyses conducted on HbA (human Hb) highlights interesting analogies (similarity of the transition pathway) and differences (distinct population of intermediate states). In particular, the ability of HbTb to significantly populate intermediate states along the functional pathway explains the observed propensity of this protein to assume these structures in the crystalline state. It also explains some functional data reported on the protein that indicate the occurrence of other functional states in addition to the canonical R and T ones. These findings are in line with the emerging idea that the classical two-state view underlying tetrameric Hb functionality is probably an oversimplification and that other structural states play important roles in these proteins. The ability of MD simulations to accurately describe the functional pathway in tetrameric Hbs suggests that this approach may be effectively applied to unravel the molecular and structural basis of Hbs exhibiting peculiar functional properties as a consequence of the environmental adaptation of the host organism.

Figure 1

Root-mean-square deviation (RMSD) analysis. RMSD values (computed on C^α atoms) of the trajectory structures versus the starting T model (black, PDB ID: 2H8F) and the R state (red, PDB ID: 1PBX) in the HbTb simulations (runs r1 to r10). Green boxes indicate simulation runs with observed T → R transition.

Figure 2

Iron–iron distances. Distances between the heme iron (Fe) atoms computed for the α1-β2 (black) and β1-β2 (red) dimers in the HbTb simulation runs with observed T→R transition. Correspondent Fe–Fe distance values detected in the crystallographic structures of the T and R state of HbTb are indicated by solid and dashed lines, respectively.

Figure 3

Essential dynamics analysis. Projection on the first eigenvector of the MD trajectories with observed T→R transition. The vertical solid lines correspond to the projections of the crystallographic structures of HbTb states: T (black, PDB ID: 2H8F) and R (red, PDB ID: 1PBX); HbTn intermediates: TnA (dark green, PDB ID: 5LFG), TnB (magenta, PDB ID: 5LFG), and TnH (cyan, PDB ID: 3D1K); HbA states: intermediate HL-(C) (violet, PDB ID: 4N7P), R2 (blue, PDB ID: 1BBB), RR2 (yellow, PDB ID: 1MKO), and R3 (orange, PDB ID: 4NI0).

Figure 4

Time evolution of the structural probes that are characteristic of the different HbTb states in the r1 simulation run. Specifically, the distances (a) N^ζ Lys40α1–OT His146β2, (b) O^η Tyr42α1–O^δ Asp99β2, (c) C^α Ser44α1–C^α His97β2, and (d) C^α His146β1–C^α His146β2 are monitored.

Figure 5

Inter-aspartic distances at α1β2 (left) and α2β1 (right) interfaces as observed along the MD trajctories. Minimum distances between Asp O^δ atoms of the pairs Asp95α1–Asp101β2 and Asp95α2–Asp101β1 are reported. Red line at 3.0 Å is given as an indicative H-bond length threshold.

Figure 6

Cartoon representation of the three-dimensional structure of HbTb tetramer in the T state (PDB ID: 2H8F). (a) Asp95α1, Asp99β2, and Asp101β2 of the catalytic triad are shown as sticks. Close-up view of the α1β2 interfacing Asp residues (b) in the X-ray starting structure (PDB ID: 2H8F) and (c) in a representative trajectory frame (r2 simulation run, t = 75.5 ns) upon T → R transition. See also Figure 5.

Figure 7

HbTb T–R transition at T = 273 K. RMSD values (computed on Cα atoms) of the trajectory structures versus the starting T model (black, PDB ID: 2H8F) and the R state (red, PDB ID: 1PBX) for (a) r1L and (b) r1L* simulations performed at 273 K. Projection of the MD trajectory on the first eigenvector for (c) r1L and (d) r1L*. The vertical solid lines correspond to the projections of the crystallographic structures of HbTb states: T (black, PDB ID: 2H8F) and R (red, PDB ID: 1PBX); HbTn intermediates: TnA (dark green, PDB ID: 5LFG), TnB (magenta, PDB ID: 5LFG), and TnH (cyan, PDB ID: 3D1K); HbA states: intermediate HL-(C) (violet, PDB ID: 4N7P), R2 (blue, PDB ID: 1BBB), RR2 (yellow, PDB ID: 1MKO), and R3 (orange, PDB ID: 4NI0).