PT - JOURNAL ARTICLE AU - Bin Sun AU - Xuan Fang AU - Christopher N. Johnson AU - Garrett Hauck AU - Jonathan P. Davis AU - Peter M. Kekenes-Huskey TI - The non-canonical interaction between calmodulin and calcineurin contributes to the differential regulation of plant-derived calmodulins on calcineurin AID - 10.1101/2021.06.18.449055 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.06.18.449055 4099 - http://biorxiv.org/content/early/2021/06/18/2021.06.18.449055.short 4100 - http://biorxiv.org/content/early/2021/06/18/2021.06.18.449055.full AB - Calmodulin (CaM) is an important Ca2+ signaling hub that regulates many protein signaling pathways. In recent years, several CaM homologs expressed in plants have been shown to regulate mammalian targets and they are attractive for gene therapy. However, the molecular basis of how the CaM homologs mutations impact target activation is unclear, which limits efforts to engineer their functional properties. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isofroms, sCaM-1 and sCaM-4 share >90% and ~ 78% identity with the mammalian CaM (mCaM), respectively, activate CaN with comparable or reduced activity relative to mCaM. We used molecular simulations and experimental assays to probe whether calcium and protein-protein binding interactions are altered in plant CaMs relative to mCaM as a basis for differential CaN regulations. We found that the two sCaMs’ Ca2+-binding properties such as coordination and affinity are comparable to mCaM. Further, the binding of CaM to the CaM binding region (CaMBR) in CaN is also comparable among the three CaMs, as evidenced by calculated binding free energies and experimental measured EC50 [CaM]. However, mCaM and sCaM-1 exhibited stronger binding with a secondary region of CaN’s regulatory domain that is weakened for sCaM-4. This secondary interaction is likely to affect the turnover rate (kcat) of CaN based on our modeling of enzyme activity and is consistent with our experimental data. Together, our data show how plant-derived CaM variants can alter target activation through interactions beyond Ca2+-binding and canonical CaMBR binding, which may extend beyond the mammalian CaN target.Competing Interest StatementThe authors have declared no competing interest.