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

Vandana Mishra,Sameeta Bilgrami,Radhey Shyam Sharma,Punit Kaur,Savita Yadav,Ruth Krauspenhaar,Christian Betzel,Wolfgang Voelter,Cherukuri R Babu,Tej P Singh

doi:10.1074/jbc.m500735200

Abstract

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.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The Overall Structure—The general organization of Himalayan mistletoe ribosome inactivating protein (HmRIP) (Fig. 4) is essentially similar to that observed for other type II Ribosome-inactivating proteins (RIPs) (34 –39)
Detection of A New, Functionally Active Sugar-binding Site in 1␤-Subdomain—We have identified the presence of a third functionally active sugar-binding site in the 1␤-subdomain of HmRIP

Summary

Introduction

The atomic coordinates and structure factors (code 1YF8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http:// www.rcsb.org/). The 1␤-site is active despite the absence of conserved polar sugar-binding residues. The AICP ligand forms several interactions with a number of key residues at the N-glycosidase active site of the toxin subunit (Fig. 5A).

Results

Conclusion