Calcium binding and induced folding of osteocalcin as a function of its carboxylation degree investigated by biophysical approaches.

Osteocalcin (OC) is a 49-amino-acids protein that plays several biological roles, from regulation of bone mineralization to biomarker of bone turnover and metabolic hormone. $In vivo$, OC undergoes vitamin-K-dependent $\gamma$-carboxylation of three glutamyl residues. In this contribution, the behaviour of the eight differently carboxylated OC forms has been investigated through biochemical and complementary biophysical approaches: circular dichroism and Fourier transform infrared spectroscopies, native mass spectrometry and isothermal titration calorimetry. In particular, we found that the carboxylation degree affects the affinity for $Ca^{2+}$ ions, the secondary structure of the free OC variants and the calcium-induced conformational transition of the proteins. Interestingly, the complementary information obtained by these biophysical approaches clearly disclosed that the three $\gamma$-carboxyglutamic acids of OC are not equivalent for calcium affinity and protein conformational features. Our data could provide a structural explanation of the very different biological roles of this multifunctional protein. This work has been supported by Fondazione Cariplo, grant No. 2018-0458.