Localization of structural elements of bee venom phospholipase A(2) involved in N-type receptor binding and neurotoxicity

We have shown previously that neurotoxic venom secretory phospholipases A(2) (sPLA(2)s) have specific receptors in brain membranes called N-type receptors that are likely to play a role in the molecular events leading to neurotoxicity of these proteins. The sPLA(2) found in honey bee venom is neurotoxic and binds to this receptor with high affinity. In this paper, we have used a number of mutants of bee venom sPLA(2) produced in Escherichia coli to determine the structural elements of this protein that are involved in its binding to N-type receptors. Mutations in the interfacial binding surface, in the Ca2+-binding loop and in the hydrophobic channel lead to a dramatic decrease in binding to N-type receptors, whereas mutations of surface residues localized in other parts of the sPLA(2) structure do not significantly modify the binding properties. Neurotoxicity experiments show that mutants with low affinity for N-type receptors are devoid of neurotoxic properties, even though some of them retain high enzymatic activity. These results provide further evidence for the involvement of N-type receptors in neurotoxic processes associated with venom sPLA(2)s and identify the surface region surrounding the hydrophobic channel of bee venom sPLA(2) as the N-type receptor recognition domain.