Evidences for structural basis of altered ascorbate peroxidase activity in cadmium-stressed rice plants exposed to jasmonate

Abstract

Binding interactions of cadmium (Cd) with rice ascorbate peroxidase (OsAPX) in presence or absence of jasmonate was examined in-silico. OsAPX is a 250 amino acid long protein with 90 % sequence similarity to soybean-APX. The 3D model of OsAPX obtained by homology modeling using soybean APX (PDBID:1OAF) as template was associated with −15975.85 kJ/mol energy, 100 % residues in favoured region, verify score of 0.85, ERRAT score 89.625 and a negative ProSA graph, suggesting OsAPX model to be of good quality, robust and reliable which was submitted with Protein Model Database with PMDBID: PM0078091. The rice ascorbate peroxidase ascorbate [OsAPX–Asc] complex had a substrate binding cavity involving residues at position ³⁰KSCAPL³⁵, ¹⁶⁷RCH¹⁶⁹ and ¹⁷²R wherein ascorbate accommodated via three H-bonds involving ³⁰Lys at the γ-edge of heme. ¹⁶⁹His served as a bridge between heme-porphyrin of OsAPX and ascorbate creating a charge relay system. Cd bound in [OsAPX–Asc–Cd] complex at ²⁹EKSCAPL³⁵, a site similar to ascorbate binding site. The binding of Cd caused breaking of ¹⁶⁹His bridge shifting the protein conformation. Cadmium exhibited four electrostatic interactions via ²⁹Glu of OsAPX backbone. Docking of [OsAPX–Asc] with jasmonic acid (JA) resulted in [OsAPX–Asc–JA] complex where 4—H-bonds held JA to OsAPX in a cavity at γ-edge on the distal side of heme. The binding of [OsAPX–Asc–JA] to Cd show the metal to bind at a position other than that involved in binding of OsAPX with Cd alone. Results indicate that Cd does not replace iron or ascorbate or JA but binds to OsAPX on the surface at a separate site electrostatically. In presence of JA the interactions involved in formation of [OsAPXAsc] are restored which is otherwise altered by the presence of Cd. The formation and reformation of H-bond take place between the [OsAPX–Asc] and Cd/JA. It is the interaction between heme and ascorbate which is modulated differently in presence of Cd/JA. In absence of JA, Cd-binds to the [OsAPX–Asc] complex at the proximal end of APX near Asc-binding site, whereas in presence of JA, Cd-binds on the opposite site of the Asc-binding site involving ³⁰Lys and ²⁹Glu residues. In-silico binding studies well correlate with the wet-lab results where exogenous application of JA increased the activity of OsAPX in rice grown under Cd-stress. Therefore it is concluded that the activity of OsAPX in rice roots and shoots are compromised under Cd-stress alone.