Probing the Role of Murine Neuroglobin CDloop-D-Helix Unit in CO Ligand Binding and Structural Dynamics

Abstract

We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop-D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop-D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop-D-helix in neuroprotective signaling in a cellular or animal model.

Figure 1

Crystal structure of the neuroglobin CDless mutant determined at a 1.80 Å resolution. (A) The structure of the CDless mutant revealed the presence of two monomers in the asymmetric unit: MON₁ in dark pink and MON₂ in dark orange. The interactions between hydrophobic patches of symmetry-related MON₁ and MON₂ are shown in the inset. The CElink (protein segment between the C- and E-helices) conformation of Ngb CDless MON₁ (dark pink in B) and MON₂ (dark orange in C) is shown in comparison to the CDloop in Ngb wild type (yellow in panels B and C). The corresponding heme insertion positions for MON₁ (B) and MON₂ (C) are represented in orange for the canonical insertion and in blue for the reversed one. 2Fo–Fc maps are shown in light blue and contoured at 1σ.

Figure 2

Heme environment in Ngb CDless. Superposition of the structure of Ngb wild type (yellow) on the structure of CDless MON₂ (A) shows the effects of the CDless mutation on the phenylalanine hydrophobic core of murine Ngb. Panels (B) and (C) show the loss of interactions between the heme propionates and the original CDloop upon the CDless mutation, respectively, in MON₁ and MON₂. The wild-type protein (pdb code 1Q1F), CDless MON₁, and MON₂ mutants are represented in yellow, pink, and orange, respectively. Canonical and reversed heme insertions are displayed in orange and blue. Water molecules belonging to the wild type and to the mutant structures are displayed as yellow and red spheres, respectively. Dashed lines correspond to H-bonds and electrostatic interactions between distal residues and the heme propionates. Red and yellow indicate Ngb CDless and wild type, respectively. Ngb CDless 2Fo–Fc maps are contoured at 1σ.

Figure 3

Effect of the CDless mutation on the heme environment probed by resonance Raman spectroscopy. (A) Comparison of the high-frequency RR spectra of the CDless mutant in solution (black line) and in crystal (dotted line) states. The spectra have been normalized to the ν₁₀ band at 1639 cm^–1. Experimental conditions: λ_exc 514.5 nm; solution: laser power at the sample 2 mW, average of six spectra with a 30 min integration time; crystal: laser power at the sample 5 μW, average of 26 spectra with a 260 min integration time. (B) Curve-fitting analysis of the v₃ region of the ferric wild type (bottom) and CDless mutant (top) obtained with the 413.1 nm excitation wavelength (for the experimental conditions, see Supporting Information Figure S4). The bandwidths are 10.5 cm^–1 for the bands at 1500.5 and 1502.5 and 8.5 cm^–1 for the band at 1506.5 cm^–1. (C) RR spectra in the low- (left) and high- (right) frequency regions of the Fe(II)–¹²CO complexes of the CDless mutant (this work), and the WT, F106A, Gly-loop/F106A, and the Gly-loop mutant Ngbs. The frequencies of the ν(Fe–C) and ν(C–O) stretching modes are labeled in red (open (A₀) and closed (A₃) forms). The spectra in panels (B) and (C) have been shifted along the ordinate axis for visualization. Experimental conditions: λ_exc 413.1 nm; CDless mutant: laser power at the sample 550 μW, average of 18–27 spectra with a 180–270 min integration time for the low- and high-frequency regions. Wild type (WT), F106A, Gly-loop/F106A, and the Gly-loop mutants (see ref (18) for details). (D) Back-bonding correlation line (black) of the ν(Fe–C) and ν(C–O) stretching frequencies of various Ngbs,^,− as reported in Supporting Information Table S3, together with the corresponding data of sperm whale Mb (swMb). The dotted lines indicate the approximate delineation between the frequency zones of the A₀, A₁, and A₃ forms. The humans Ngb and swMb show also a third weak H-bonded conformer (A₁) at 505/1956 and 508/1946 cm^–1, respectively.

Figure 4

CO (re)binding kinetics of neuroglobin wild type (gray) and CDless (orange) at 25 °C. (A) Rapid mixing kinetic traces for Ngb wild type and CDless were fitted as mono- or biexponentials and corresponding rate constants k_obs. The dependence of rate constants on CO concentration is fitted (full lines) according to Hargrove and collaborators; the values are reported in Supporting Information Table S7. (B) The fraction Y of Ngb bound to CO was extracted from the overall amplitude of the kinetic traces, and log 10(Y/1-Y) was plotted as a function of the CO concentration. Data were linearly fitted to determine the overall CO affinity c₅₀. (C) CO rebinding kinetics was followed by laser flash photolysis at 25 °C at 0.2 atm CO. Data are reported as the progress curve representing the fraction of deoxy molecules, N(t), as a function of time after photolysis. Fitting curves using exponential decay functions are superimposed to the experimental data (black dots) and colored, respectively, in gray and orange for Ngb wild type and CDless. The inset is a close-up view of the kinetic trace at an early time range.

Figure 5

Molecular dynamics simulations of Ngb CDless. (A) Root-mean-square fluctuations of Cα atoms from the cumulative simulations of MON1 and MON2 in crystallo. Secondary structures and helices numbering are represented in black on top of the graph. (B) Influence of the heme insertion mode on the C-helix stability in Ngb CDless in solution. Top panel: secondary structure adopted by the C-helix-CElink in the presence of the reversed heme insertion. Bottom panel: in the presence of the canonical one. Helices are represented in blue, coils are in white, turns are in yellow, and bends are in green. (C) Close-up view of the C-helix-CElink in the presence of either the reversed (top) or the canonical heme insertion (bottom). Porphyrin groups are displayed in blue (reversed heme insertion) or orange (canonical heme insertion). (D) Free energy associated with the His64 displacement in Ngb CDless embedding either the reversed or the canonical heme.