STRUCTURE AND THERMODYNAMICS OF ADENINE BINDING TO THE RICIN A-CHAIN

Abstract

Ricin is a type II ribosome inactivating protein from the castor bean that presents a significant bioterrorism threat. Due to its high availability and toxicity, there is a need for the development of therapeutics against the toxin. The ricin A-chain (RTA) is an N- glycosidase that cleaves a specific adenine base from a conserved loop in 28S rRNA. Free adenine is an uncompetitive inhibitor of this reaction. Using a combination of fluorescence and X-ray crystallography, I investigated the energetics and active site conformational changes that occur with adenine binding to RTA. I solved an improved RTA-adenine co-crystal structure diffracting to a resolution of 1.94 A, which shows different molecular interactions than previous structures. I also examined the effects of temperature 5-30°C and pH 4-8 on purine binding to RTA. The Kᴅ for adenine binding to RTA was 7.0 x 10ˉ⁴ M at pH 7.4, 25 °C.