A calorimetric and structural analysis of cooperativity in the thermal unfolding of the PDZ tandem of human Syntenin-1.

Syntenin-1 is a multidomain protein containing a central tandem of two PDZ domains flanked by two unnamed domains. Previous structural and biophysical studies show that the two PDZ domains are functional both isolated and in tandem, occurring a gain in their respective binding affinities when joined through its natural short linker. To get insight into the molecular and energetic reasons of such a gain, here, the first thermodynamic characterization of the conformational equilibrium of Syntenin-1 is presented, with special focus on its PDZ domains. These studies include the thermal unfolding of the whole protein, the PDZ-tandem construct and the two isolated PDZ domains using circular dichroism, differential scanning fluorimetry and differential scanning calorimetry. The isolated PDZ domains show low stability (ΔG < 10 kJ·mol) and poor cooperativity compared to the PDZ-tandem, which shows higher stability (20-30 kJ·mol) and a fully cooperative behaviour, with energetics similar to that previously described for archetypical PDZ domains. The high-resolution structures suggest that this remarkable increase in cooperativity is associated to strong, water-mediated, interactions at the interface between the PDZ domains, associated to nine conserved hydration regions. The low T value (45 °C), the anomalously high unfolding enthalpy (>400 kJ·mol), and native heat capacity values (above 40 kJ·K·mol), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics.