Electrostatic Tuning of the Ligand Binding Mechanism by Glu27 in Nitrophorin 7

Abstract

Nitrophorins (NP) 1-7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

Figure 1

Key structural features of NP7. (A) Representation of the X-ray structure of NP7 from Rhodnius prolixus (PDB entry 4XME) showing the coordination of the heme to H60, the location of the residues (D32, I132) that form the hydrogen bond implicated in the conformational transition between open and closed states, and the position of E27, which is replaced by valine in other nitrophorins. Loops AB, EF, and GH are shown in magenta, blue, and orange, respectively. The hydrogen bond between E27 and T168 (distance: 2.8 Å) is shown as a dashed line. (B) Surface representation of NP7. Rear view opposite to the mouth of the heme cavity. Lysine residues are shown in blue.

Figure 2

Ligand rebinding kinetics. (A) Time-dependent transient absorption spectra for CO-bound NP7(E27V) following femtosecond photoexcitation at 530 nm at 4 (black line), 200 (red line), 400 (green line) and 800 ps (blue line) delay times. First spectral component (U₁S₁ on a 1:10 scale) obtained from the SVD analysis of the time resolved differential absorption spectra multiplied by the corresponding singular value (S₁ = 40.5) is shown as the cyan line. (B) Comparison between the time course of the amplitude V₁ (black open circles) of the main spectral component obtained from SVD for CO-bound NP7 (E27V), and the normalized transient absorbance at 436 nm as a function of the delay time (black line). pH = 7.5, T = 20 °C. The amplitude V₁ of the main spectral component obtained from SVD at pH 5.5 is reported as the red solid circles. Complete rebinding kinetics to (C) NP7(E27V) and (D) NP7(E27Q) at pH = 7.5 (green) and pH = 5.5 (black) reported as fraction of unliganded molecules vs time. The experimental progress curves were recorded at 1 CO atm (filled circles) and 0.1 CO atm (open circles). For comparison the data for CO rebinding to wt NP7 are reported in plots C and D at 1 atm CO at pH 5.5 (blue circles) and 7.5 (cyan circles).

Figure 3

Minimal reaction scheme for the observed rebinding kinetics.

Figure 4

X-ray structure of Np7(E27V). Superposition of the X-ray structure of wt NP7 (PDB entry 4XMC; grey) and the aquo form of NP7(E27V) (PDB entry 5M6J; cyan). (B) Electron density maps (2Fo-Fc; contoured at 1σ) of the heme in (left) NP7(E27V) and (right) NP7(E27V)ImH. (C) Local structural details of the heme pocket in NP7(E27V) (cyan) and NP7(E27V)ImH (yellow) after superposition of the protein backbone. Selected residues and water molecules are shown as sticks and spheres, respectively. The network of hydrogen bonds is shown as dashed lines.

Figure 5

Structural analysis of simulated proteins. (A) Superposition of the energy-minimized averaged structures collected from the last 20 ns of MD simulations run for the closed forms of wt NP7 (green) and the E27V (A- and B-heme; yellow) and E27Q (Q1 and Q2; orange) variants. The heme group is shown as sticks. (B,C) RMSF (Å) profile by residue (excluding hydrogen atoms) for wt NP7 (dashed black) and its mutated E27V (B-heme: green; A-heme: red) and E27Q (Q1: green; Q2: red) variants. (D) Position of the heme and selected residues (Glu27, Thr168, and Tyr30) after superposition of the protein backbone in the X-ray and MD energy-minimized averaged structures of wt NP7 and its E27V and E27Q mutated variants. Hydrogen bonds are shown as dashed lines.

Figure 6

Thermodynamic cycle that relates the ionization state of Asp32 in NP7 and Asp30 in NP4 with the conformational transition between open and closed states of the proteins. The protonated and ionized forms of Asp32/Asp30 are indicated with the superscripts 0 and −, and the open and closed conformations are denoted with the superscripts O and C. K_a^o and K_a^C denote the ionization constants of Asp32/Asp30 in the open and closed states, and K_D and K_H stand for the conformational equilibrium constant in the dissociated and protonated forms. The pK_a^o and pK_a^C values were determined from APBS computations (the pKa values reported in ref. are given in parenthesis).

Figure 7

Dependence of the apparent pKa of wt NP4 and NP7, and the mutated variants NP7(E27V) and NP7(E27Q) on the equilibrium constant (K_D) for the conversion between the closed and open forms of the ionized protein.