Equilibrium unfolding of the PDZ domain of β2-syntrophin.
β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of β2S-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 the regulation of insulin secretion.
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina.
Gabriela María
GM
Torchio
Mario Roberto
MR
Ermácora
Mauricio Pablo
MP
Sica
D000595:Amino Acid Sequence; D049029:Dystrophin-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D054731:PDZ Domains; D011489:Protein Denaturation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D049029:Dystrophin-Associated Proteins; D007328:Insulin; C091824:syntrophin; D014508:Urea