Abstract
Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic RNase A family. Although its physiological function is still unclear, it has been shown that EDN is a neurotoxin capable of inducing the Gordon phenomenon in rabbits. EDN is also a potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now determined high resolution (1.8 A) crystal structures for EDN in complex with adenosine-3',5'-diphosphate (3',5'-ADP), adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate refined at 1.6 A. The inhibition constant of these mononucleotides for EDN has been determined. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They also provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN.
Highlights
Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic ribonuclease A (RNase A) family
In the EDN1⁄73Ј,5Ј-ADP complex, the 5Ј-phosphate binds at the P1 site engaging in similar interactions with EDN to those observed for phosphate groups bound to RNase A subsite P1
The adenosine binds in an identical manner to that observed for adenosines bound to the subsite B2 of RNase A
Summary
Vol 276, No 18, Issue of May 4, pp. 15009 –15017, 2001 Printed in U.S.A. HIGH RESOLUTION CRYSTAL STRUCTURES OF EDN COMPLEXES WITH ADENYLIC NUCLEOTIDE INHIBITORS*. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN. Two sulfate anions were found in the EDN1⁄7sulfate complex structure (13) occupying two distinct subsites One of these is subsite P1, whereas the other is a site not identified in the structure of RNase A, suggested to correspond to a new PϪ1 phosphate-binding subsite. The structures of the complexes have revealed a detailed picture of critical residues involved in the P1 and B2 substrate-binding sites and their flexibility in interaction with different adenylic nucleotides.
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