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

Jose C Martinez; Javier Ruiz-Sanz; María J Resina; Fernando Montero; Ana Camara-Artigas; Irene Luque

doi:10.1016/j.ijbiomac.2023.124662

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

Int J Biol Macromol. 2023 Jul 1;242(Pt 1):124662. doi: 10.1016/j.ijbiomac.2023.124662. Epub 2023 Apr 27.

Authors

Jose C Martinez¹, Javier Ruiz-Sanz², María J Resina³, Fernando Montero⁴, Ana Camara-Artigas⁵, Irene Luque⁶

Affiliations

¹ Department of Physical Chemistry, Institute of Biotechnology and Excellence Unit in Chemistry Applied to Biomedicine and Environment, Faculty of Sciences, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain. Electronic address: jcmh@ugr.es.
² Department of Physical Chemistry, Institute of Biotechnology and Excellence Unit in Chemistry Applied to Biomedicine and Environment, Faculty of Sciences, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain. Electronic address: jruizs@ugr.es.
³ Department of Physical Chemistry, Institute of Biotechnology and Excellence Unit in Chemistry Applied to Biomedicine and Environment, Faculty of Sciences, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain. Electronic address: mjresina@ugr.es.
⁴ Department of Physical Chemistry, Institute of Biotechnology and Excellence Unit in Chemistry Applied to Biomedicine and Environment, Faculty of Sciences, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain. Electronic address: fmontero@ugr.es.
⁵ Department of Chemistry and Physics, Agrifood Campus of International Excellence (ceiA3) and CIAMBITAL, University of Almería, Carretera de Sacramento s/n, 04120 Almería, Spain. Electronic address: acamara@ual.es.
⁶ Department of Physical Chemistry, Institute of Biotechnology and Excellence Unit in Chemistry Applied to Biomedicine and Environment, Faculty of Sciences, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain. Electronic address: iluque@ugr.es.

PMID: 37119899
DOI: 10.1016/j.ijbiomac.2023.124662

Abstract

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^-1) and poor cooperativity compared to the PDZ-tandem, which shows higher stability (20-30 kJ·mol^-1) 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_m value (45 °C), the anomalously high unfolding enthalpy (>400 kJ·mol^-1), and native heat capacity values (above 40 kJ·K^-1·mol^-1), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics.

Keywords: Domains in tandem; Folding thermodynamics; Protein hydration.

MeSH terms

Calorimetry
Calorimetry, Differential Scanning
Circular Dichroism
Humans
Protein Denaturation
Protein Folding*
Syntenins*
Thermodynamics

Substances

Syntenins