Crystallization of neuropilin domains and their complexes with semaphorin and VEGF

Abstract

Neuropilin-1 (Np-1) and Neuropilin-2 (Np-2) were first identified as a Semaphorin receptor involved in neuron guidance. Subsequent studies demonstrated that Np-1 and Np-2 also bind an isoform of vascular endotherial growth factor (VEGF) homologs, suggesting that Np-1 and Np-2 may also function in angiogenesis. We overproduced and purified various neuropilin domains in Hi5 insect cells using recombinant baculoviruses. Through in vitro binding experiments, we confirmed the interactions between VEGF165 and neuropilin b1/b2 domain, Semaphorin3A and neuropilin a1/a2 domain. We also report previously unidentified interactions between Nps and Flt-1 by ion-exchange chromatography and gel filtration. The neuropilin a1/a2 domain and complexes with Semaphorin3A were crystallized by vapor diffusion technique. The crystal structure of Nps complex containing VEGF165 or Semaphorin3A will be used as a template for the design of novel drugs against neovascular and neuronal diseases.