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Crystal Structure of Himalayan Mistletoe Ribosome-inactivating Protein Reveals the Presence of a Natural Inhibitor and a New Functionally Active Sugar-binding Site

2005; Elsevier BV; Volume: 280; Issue: 21 Linguagem: Inglês

10.1074/jbc.m500735200

1083-351X

Vandana Mishra, S. Bilgrami, Radhey Shyam Sharma, Punit Kaur, Savita Yadav, R. Krauspenhaar, Christian Betzel, Wolfgang Voelter, Cherukuri R. Babu, T.P. Singh,

Plant Virus Research Studies

Ribosome-inactivating proteins (RIPs) are toxins involved in plant defense. How the plant prevents autotoxicity is not yet fully understood. The present study is the first structural evidence of a naturally inhibited form of RIP from a plant. Himalayan mistletoe RIP (HmRIP) was purified from Viscum album leaves and crystallized with lactose. The structure was determined by the molecular replacement method and refined at 2.8-A resolution. The crystal structure revealed the presence of high quality non-protein electron density at the active site, into which a pteridine derivative (2-amino 4-isopropyl 6-carboxyl pteridine) was modeled. The carboxyl group of the ligand binds strongly with the key active site residue Arg(162), nullifies the positive charge required for catalysis, and thereby acts as a natural inhibitor. Lectin subunits of RIPs have two active sugar-binding sites present in 1alpha- and 2gamma-subdomains. A third functionally active site has been identified in the 1beta-subdomain of HmRIP. The 1beta-site is active despite the absence of conserved polar sugar-binding residues. Loss of these residues is compensated by the following: (i) the presence of an extended site where the penultimate sugar also interacts with the protein; (ii) the interactions of galactose with the protein main chain carbonyl and amide nitrogen atoms; (iii) the presence of a well defined pocket encircled by four walls; and (iv) a favorable stacking of the galactose ring with Tyr(66) besides the conserved Phe(75). The mode of sugar binding is also distinct at the 1alpha and 2gamma sugar-binding sites.