Equilibrium unfolding of the PDZ domain of β2-syntrophin

Abstract

b2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic b-cells. Itcontains a PDZ domain (b2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin gran-ules to actin filaments. The phosphorylation state of b2-syntrophin allosterically regulates the affinity of b2S-PDZ for ICA512,and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfold-ing of b2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, b2S-PDZ is margin-ally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit revealsa significant unfolded fraction under physiological conditions. Furthermore, Tm and Tmax denaturant-dependent shifts andnoncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail toexplain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 andthe results of molecular dynamics simulations indicate that this behavior of b2S-PDZ might be related to its charge distribution.All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and theregulation of insulin secretion.