Sensing the allosteric force

Abstract

Allosteric regulation is an innate control in most metabolic and signalling cascades that enables living organisms to adapt to the changing environment by tuning the affinity and regulating the activity of target proteins. For a microscopic understanding of this process, a protein system has been designed in such a way that allosteric communication between the binding and allosteric site can be observed in both directions. To that end, an azobenzene-derived photoswitch has been linked to the α3-helix of the PDZ3 domain, arguably the smallest allosteric protein with a clearly identifiable binding and allosteric site. Photo-induced trans-to-cis isomerisation of the photoswitch increases the binding affinity of a small peptide ligand to the protein up to 120-fold, depending on temperature. At the same time, ligand binding speeds up the thermal cis-to-trans back-isomerisation rate of the photoswitch. Based on the energetics of the four states of the system (cis vs trans and ligand-bound vs free), the concept of an allosteric force is introduced, which can be used to drive chemical reactions.

Fig. 1. Schematic display of the photocontrollable PDZ3 system complexed with the KETWV peptide.

The left panel shows the cis configuration, which promotes the α3 helix, and the right panel the trans configuration, which disturbs it. The cysteine residues chosen as anchoring points for the photoswitch are shown in pink. The ligand-bound structure has been adapted from an X-ray structure PDB:1tp5, which is for a somewhat longer peptide (KKETWV) bound to PDZ3, by removing one N-terminal lysine to reveal the peptide ligand used here.

Fig. 2. Spectroscopic observables.

a CD spectra of the photocontrollable PDZ3 domain in trans (green) and cis (violet), showing the difference in helicity between the two configurations of the photoswitch. Inlet graph shows the trans to cis CD difference spectrum, which corresponds to the CD signal of an isolated helical protein. b Fraction of bound ligand as a function of initial protein concentration [P₀] with the initial ligand concentration [L₀] kept fixed at 15 μM. Data are shown for the photoswitch in trans (green) and cis (violet) configurations and at 10 °C (top) and 40 °C (bottom). c Thermal cis-to-trans relaxation of the photocontrollable PDZ3 alone (red), and with saturating concentrations of the ligand (blue) at 10 °C (top) and 40 °C (bottom). Source data are provided as a Source Data file.

Fig. 3. Energetics.

a Van-t’Hoff plots of the binding affinity of the peptide ligand to the photoswitchable PDZ3 domain in its cis (magenta) and trans-state (green). The binding enthalpies ΔH and entropies ΔS extracted from the linear fits are indicated. b Arrhenius plots of the thermal cis-to-trans isomerisation rate for the free protein (P, in red) or with the ligand bound (PL, in blue). The activation enthalpies ΔH^# and the pre-exponential factors A are indicated. All data are also listed in Table 1, and the corresponding titration and relaxation measurements are shown in SI as Figs. S6 and S7. Error bars, which have been estimated from the fits of the data shown in Figs. S6 and S7, indicate single standard deviation around the mean. Source data are provided as a Source Data file.

Fig. 4. Thermodynamic cycles.

a Conventional thermodynamic cycle of the two-state model of allostery with P and P* being resting and active states of the protein, and P + L and PL the apo and ligand-bound states, respectively. In contrast, b the thermodynamic cycle of the photoswitchable system, where the cis-to-trans reaction is essentially unidirectional, but can transiently be put out of equilibrium by light, as indicated by the flash and the curved dotted arrow. c Energy profiles (not on scale) of the thermal cis-to-trans isomerisation of the free protein P (red) or the ligand bound protein PL (blue).