Structural Biology

Biography

Biography: Alessandra Astegno

Abstract

Calcium is an essential second messenger in plants that regulates various signaling pathways through stimulus-specific Ca²⁺ signatures, which are decoded and converted into a wide variety of biochemical changes by Ca²⁺ sensors. Besides evolutionarily conserved calmodulin (CaM), plants exclusively possess a group of calmodulin-like proteins (CMLs), which play central roles in the coordination of plant responses to different external stimuli. Nevertheless, only few of these proteins have been thoroughly characterized and demonstrated to function as Ca²⁺ sensors.

Our research is focused on the investigation of the metal-binding, physicochemical and structural properties of various plant CMLs using complementary biophysical and structural approaches in order to correlate their properties  with the biological activity.

We have recently characterized CML36 from Arabidopsis thaliana, demonstrating that in vitro the protein  shows features consistent with  Ca²⁺ sensor function. ITC analysis revealed that CML36 possesses two high affinity Ca²⁺/Mg²⁺ mixed sites and two low affinity Ca²⁺-specific sites. Binding of Ca²⁺ to CML36 increases its α-helical content and triggers a conformational change that exposes hydrophobic surfaces necessary for target recognition. Ca²⁺ and Mg²⁺ ions also stabilized the tertiary structure of CML36. In particular, cations binding to the Ca²⁺/Mg²⁺ mixed sites appears to guide a large structural transition from a loosely packed molten globule apo-state to a well-defined, stable holo-structure. Through in vitro binding experiments, we showed that CML36 directly interacts with the N-terminal domain of Arabidopsis Ca²⁺-ATPase isoform 8 (ACA8), a type IIB Ca²⁺ pump localized at the plasma membrane (PM). Moreover, we demonstrated that this interaction promotes ACA8 Ca²⁺-dependent hydrolytic activity in vitro.

References:

1. Astegno A, Maresi E, Bertoldi M, La Verde V, Paiardini A, Dominici P (2017) Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii. Biochem J. 474(6):939-955.
2. Vallone R, La Verde V, D'Onofrio M, Giorgetti A, Dominici P, Astegno A (2016) Metal  binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana. Protein Sci. 25(8):1461-71.
3. Kumar N, Astegno A, Chen J, Giorgetti A, Dominici P (2016) Residues in the distal heme pocket of Arabidopsis non-symbiotic hemoglobins: implication for nitrite reductase activity. Int J Mol Sci. 28;17(5).
4. Astegno A, La Verde V, Marino V, Dell'Orco D, Dominici P (2016) Biochemical and biophysical characterization of a plant calmodulin: Role of the N- and C-lobes in calcium binding, conformational change, and target interaction. Biochim Biophys Acta. 1864(3):297-307.
5. Astegno A, Capitani G, Dominici P (2015) Functional roles of the hexamer organization of plant glutamate decarboxylase. Biochim Biophys Acta. 1854(9):1229-37.