A simple method to determine changes in the affinity between HisF and HisH in the Imidazole Glycerol Phosphate Synthase heterodimer

Abstract

The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (Kd). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the Kd at four different temperatures. These Kd values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the Kd determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the Kd caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the Kd, can be carried out using common laboratory equipment and is scalable.

Fig 1. Interface of the HisF-HisH heterodimer showing the buried residue hW123.

HisH is shown in cyan and HisF in green. A) Structure of the HisF-HisH heterodimer (PDB 1GPW) evidencing a single tryptophan residue (hW123 and fW156; in red) in both proteins. B) HisH has an exposed tryptophan residue (hW123) that is buried in the molecular surface of HisF residues in the heterodimer interface (green). Structures were visualized using Pymol Viewer.

Fig 2. Effect of the dimerization on the HisF and HisH intrinsic fluorescence.

The normalized spectra of HisF (red), HisH (blue) and HisF-HisH heterodimer (purple) present maximum fluorescence intensities at 321, 341 and 326 nm, respectively.

Fig 3. Intrinsic fluorescence spectra used in the determination of the dissociation constant of the HisF-HisH heterodimer.

A) Fluorescence spectra of different concentrations of HisF. B) Fluorescence spectra collected along the mixing of 15 μM HisH with sub-stoichiometric HisF concentrations (1 to 5 μM). C) Arithmetical addition spectra combining the fluorescence of HisH (from B) with the fluorescence of different HisF concentrations (from A). D) Difference spectra calculated based on the arithmetical subtraction of the “addition spectra” (C) from the “mixing spectra” (B).

Fig 4. Determination of the dissociation constant of the HisF-HisH heterodimer at different temperatures.

A) Effect of the initial HisF concentration ([F₀]) on the HisF-HisH heterodimer concentration in the equilibrium ([FH]). Fluorescence readings at 321 nm were used as proxy for the HisF and the heterodimer concentration. Those data were extracted from Fig 3A and 3C, respectively. The linear correlation coefficients (R²) are 0.96, 0.99, 0.99 and 0.98 at 30°C, 40°C, 50°C and 60°C, respectively. The slopes are 0.29, 0.37, 0.42 and 0.53 at 30°C, 40°C, 50°C and 60°C, respectively. B) Effect of the temperature on the dissociation constant of the HisF-HisH heterodimer. The K_d were calculated based on the slopes of the lines shown in panel A. More details on Material and methods.

Fig 5. Determination of the dissociation constant of the HisF-HisH heterodimer at different temperatures using ITC.

Fig 6. Thermodynamic parameters of the HisF and HisH binding calculated from the ITC experiments at different temperatures.

Fig 7. Correlation between K_d of the HisF-HisH heterodimer determined using the intrinsic fluorescence and ITC methods.

Fig 8. Effect of HisF mutations on the dissociation constant (K_d) of the HisF-HisH heterodimer.

Experiments were performed at 30°C. Error bars were based on the relative deviation of the dissociation constant in the experiments using the wild-type HisF (n = 6). Mutant HisF subunits are identified by the single residue exchange present on them.