Single-Molecule Folding Mechanism of an EF-Hand Neuronal Calcium Sensor

• Published in: Structure (London) 2013-10, Vol.21 (10), p.1812-1821
• Main Authors: Heidarsson, Pétur O., Otazo, Mariela R., Bellucci, Luca, Mossa, Alessandro, Imparato, Alberto, Paci, Emanuele, Corni, Stefano, Di Felice, Rosa, Kragelund, Birthe B., Cecconi, Ciro
• Format: Article
• Language: English
• Subjects: Binding Sites; Calcium - chemistry; Humans; Molecular Dynamics Simulation; Neuronal Calcium-Sensor Proteins - chemistry; Neuropeptides - chemistry; Optical Tweezers; Protein Binding; Protein Refolding; Protein Structure, Secondary
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2013.07.022

EF-hand calcium sensors respond structurally to changes in intracellular Ca2+ concentration, triggering diverse cellular responses and resulting in broad interactomes. Despite impressive advances in decoding their structure-function relationships, the folding mechanism of neuronal calcium sensors is still elusive. We used single-molecule optical tweezers to study the folding mechanism of the human neuronal calcium sensor 1 (NCS1). Two intermediate structures induced by Ca2+ binding to the EF-hands were observed during refolding. The complete folding of the C domain is obligatory for the folding of the N domain, showing striking interdomain dependence. Molecular dynamics results reveal the atomistic details of the unfolding process and rationalize the different domain stabilities during mechanical unfolding. Through constant-force experiments and hidden Markov model analysis, the free energy landscape of the protein was reconstructed. Our results emphasize that NCS1 has evolved a remarkable complex interdomain cooperativity and a fundamentally different folding mechanism compared to structurally related proteins. •The folding of NCS1 was studied with optical tweezers and molecular dynamics•The two NCS1 structural domains exhibited striking interdomain cooperativity•NCS1 folding is synchronized to Ca2+-ion binding into the three EF-hands•The free energy landscape was reconstructed using hidden Markov model analysis Neuronal Ca2+ sensors respond structurally to changes in intracellular Ca2+ concentrations, yet the details of their conformational dynamics are largely uncharacterized. Heidarsson et al. use a single-molecule study of NCS1 to describe the complex folding network and reconstruct the energy landscape of the protein.