Crystal structure of himalayan mistletoe ribosome-inactivating protein reveals the presence of a natural inhibitor and a new functionally active sugar-binding site.
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.
Mishra
Vandana
V
Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110 029, India.
Bilgrami
Sameeta
S
Sharma
Radhey Shyam
RS
Kaur
Punit
P
Yadav
Savita
S
Krauspenhaar
Ruth
R
Betzel
Christian
C
Voelter
Wolfgang
W
Babu
Cherukuri R
CR
Singh
Tej P
TP
D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D017124:Conserved Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D018515:Plant Leaves / Q000737:chemistry; D028321:Plant Preparations / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011621:Pteridines / Q000737:chemistry; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological / Q000737:chemistry* / Q000378:metabolism; D028181:Viscum / Q000737:chemistry*
D028321:Plant Preparations; D010940:Plant Proteins; D011621:Pteridines; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum; D001120:Arginine; D007785:Lactose