Insight into the allosteric mechanism of Scapharca dimeric hemoglobin

Abstract

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

Figure 1

Crystal structures of the unliganded and CO-bound HbI. The HbI homodimer subunits are colored dark and light gray. Purple sticks represent the prosthetic heme group. Iron is colored green. CO is colored blue. The structures of unliganded HbI (left) and CO-HbI (right) are generated from the PDB entries 4SDH and 3SDH, respectively. Interfacial helices E and F are shown at the bottom, to highlight the conformational transition undergone by Phe 97 (red) upon CO binding. The structural representation was drawn using VMD.

Figure 2

Population distribution of dihedral angle χ₁ of residue 97. Shown are the distributions of the populations of dihedral angle χ₁, defined by the C, C_α, C_β, and C_γ atoms of residue 97, calculated from the MD simulations of WT HbI (a) and F97Y HbI (b). The histograms calculated from the MD trajectories of the unliganded and liganded proteins are colored white and black, respectively.

Figure 3

Comparison of the experimentally determined order parameters, S², with those calculated from the MD simulations. The S² values are shown as a function of the protein sequence for WT CO-bound HbI (a), unliganded WT HbI (c), CO-bound F97Y HbI (e), and unliganded F97Y HbI (g). Experimentally determined S² values are shown as empty circles, and S² values calculated from the MD simulations are shown as filled circles. The difference between the simulated and experimental S² (ΔS² = S_MD² – S_exp²) is shown as a function of residue number for WT CO-bound HbI (b), unliganded WT HbI (d), CO-bound F97Y HbI (f), and unliganded F97Y HbI (h). Secondary structural elements are shown at the top of each plot. Solid bars represent α-helices, and lines represent loops. Helix C and the CD loop are highlighted with a red box.

Figure 4

Difference in the order parameter between the free and bound states calculated from the MD simulations. The top panel shows ΔS² = S_CO² – S_free² calculated for each residue of WT and F97Y HbI mapped onto the respective structure. The structure of WT HbI is shown on the left and that of F97Y HbI on the right. The color scale goes from red (ΔS² = −0.005) to white (ΔS² = 0) to blue (ΔS² = 0.005). Residues characterized by a ΔS² value of ≤−0.005 are colored red, and residues characterized by a ΔS² value of ≥0.005 are colored blue. Proline residues and residues located on helix C and on the CD loop are colored gray. The structural representation was drawn using PyMOL. The bottom panel shows the difference in the order parameter between the liganded and unliganded states, averaged for each secondary structural element (ΔS² = ⟨S_CO² – S_free²⟩) between CO-bound and free WT HbI (black) and F97Y HbI (red).

Figure 5

Root-mean-square (rms) fluctuations calculated for the α-helices of HbI from the MD simulations. The rms fluctuations of each α-helix of the protein are colored black for WT HbI and red for F97Y-HbI; opaque and transparent bars are used for the liganded and unliganded states, respectively. The backbone N, C, and O atoms were used to calculate the rms fluctuations. The error bars were calculated from the standard deviation among the trajectories.

Figure 6

Secondary structure probability. The probability of being in an α-helix is shown for each residue of HbI as a function of the protein sequence. The probabilities calculated for each residue from the MD trajectories of the unliganded and liganded protein are shown with empty and filled circles, respectively. The top plot shows the results calculated from the MD simulation of WT HbI and the bottom plot those of F97Y HbI. Secondary structural elements are shown at the top of each plot. Solid bars represent α-helices, and lines represent loops.

Figure 7

Change in conformational entropy associated with CO binding calculated for each α-helix of HbI from the MD simulations. Conformational entropic contribution to the binding free energy of carbon monoxide, TΔS, estimated for each helix for WT HbI (black) and F97Y HbI (red).

Figure 8

Backbone order parameters (S²) of HbI and F97Y HbI. Order parameters, S², are shown as a function of residue number for WT HbI (a) and F97Y HbI (b). S² values calculated for CO-bound HbI are shown as filled circles, and S² values for unliganded HbI are shown as empty circles. Secondary structural elements are shown at the top of each plot. Solid bars represent α-helices, and lines represent loops.

Figure 9

Chemical exchange contributions determined from the Lipari–Szabo model-free analysis of the ¹⁵N spin relaxation measurement parameters of HbI. Residues with non-zero chemical exchange contributions, R_ex, determined from ¹⁵N spin relaxation data, are mapped in red on the structure of the protein for CO-bound HbI (a) and unliganded HbI (b). The structural representation was drawn using VMD.R_ex is shown as a function of protein sequence with filled bars and empty bars for CO-bound HbI and unliganded HbI, respectively (c). Secondary structural elements are shown at the top of the plot. Solid bars represent α-helices, and lines represent loops.