Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis

Abstract

Ophiobolin A, a fungal toxin that affects maize and rice, has previously been shown to inhibit calmodulin by reacting with the lysine (Lys) residues in the calmodulin. In the present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin molecule by site-directed mutagenesis, either by deleting them or by changing them to glutamine or arginine. We found that each of these three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding site and is responsible for all of the inhibition of ophiobolin A. When Lys-75 was removed, Lys-77 could then react with ophiobolin A to produce inhibition. Lys-148 was shown to be a binding site but not an inhibition site. The Lys-75 mutants were partially resistant to ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues were mutated, the resulting calmodulins were very resistant to ophiobolin A. Furthermore, Lys residues added in positions 86 and/or 143 (which are highly conserved in plant calmodulins) did not react with ophiobolin A. None of the mutations seemed to affect the properties of calmodulin. These results show that ophiobolin A reacts quite specifically with calmodulin.

Ophiobolin A, a sesterpenoid fungal phytotoxin, displays different mechanisms of cell death in mammalian cells depending upon the cancer cell origin

“…The sesterterpene-type compound (C25) is one of a group of over 25 Ophiobolin analogues (13), which are characterized by the unique 5-8-5 carbotricyclic skeleton (Fig. 1).…”

“…The phytotoxin is also known to bind and irreversibly antagonize calmodulin and blocks the activation of calmodulin-dependent phosphodiesterase with an IC 50 value of 9 µ M (14) by binding covalently to conserved lysine residues (13,15). However, while the mode of action has been well studied in plants, the interaction of OphA with mammalian cells is not well understood.…”

A Brief Review on Structure-Bioactivity Relationship Study of Ophiobolins and Derivatives

“…In the cytotoxic activity assay towards CLL cells, ophiobolin A, B, C, and K showed the strongest effects with LC 50 values between 1 and 8 nM, while some derivatives were inactive, ophiobolin K and 6-epiophiobolin K showed effects with LC 50 of 4 nM and Inactive ( Table 2) [3] . In the antifungal and antimicrobial activities assay, ophiobolin L showed better activity than 6-epi-ophiobolin L against most of fungi except for the Staphylococcus aureus [46] . Ophiobolin K displayed an IC 50 of 0.51 μM and 6-epi-ophiobolin an IC 50 of 2.97 μM against the both tumoral cells TK-10 and MCF-7, respectively [14] .…”

“…In vitro growth-inhibitory assays on four cancer cell lines (A549, SKMEL28, Hs683 and B16F10), the mean IC 50 growth inhibitory concentrations for 3-anhydro-6-epi-ophiobolin A were approximately 60 times higher than those for ophiobolin A [20] . Ophiobolin A can mutate Lys-75, Lys-77, and Lys-148 residues in calmodulin molecule by site-directed mutagenesis, while Lys residues in positions 86 and/or 143 which are highly conserved in plant calmodulins did not react with ophiobolin A [46] . Furthermore, kinetic analysis provide that lysine 75 in calmodulin is the primary inhibitory site for ophiobolin A [47] .…”

Ophiobolin A from Bipolaris oryzae Perturbs Motility and Membrane Integrities of Porcine Sperm and Induces Cell Death on Mammalian Somatic Cell Lines

“…These compounds show broad spectrum of biological activities, such as antimicrobial, nematocidic, cytotoxic or HIV-1 integrase inhibitory effects [9,14]. The most extensively studied representative of ophiobolins is OPA (Figure 1), the calmodulin inhibitory effect of which has been comprehensively characterized [9,15,16]. Significant reduction in the amount of different viable cells was observed after OPA treatment in ovarian cancer cell line (OVCAR3), human umbilical vein endothelial cells (HUVECs) [17] and induction of apoptotic cell death was also examined in L1210 cell line [18].…”