Crystal Structures of Eosinophil-Derived Neurotoxin (EDN) in Complex with the Inhibitors 5‘-ATP, Ap₃A, Ap₄A, and Ap₅A^,

Abstract

Eosinophil-derived neurotoxin (EDN) is a catalytically proficient member of the pancreatic ribonuclease superfamily secreted along with other eosinophil granule proteins during innate host defense responses and various eosinophil-related inflammatory and allergic diseases. The ribonucleolytic activity of EDN is central to its antiviral and neurotoxic activities and possibly to other facets of its biological activity. To probe the importance of this enzymatic activity further, specific inhibitors will be of great aid. Derivatives of 5'-ADP are among the most potent inhibitors currently known. Here, we use X-ray crystallography to investigate the binding of four natural nucleotides containing this moiety. 5'-ATP binds in two alternative orientations, one occupying the B2 subsite in a conventional manner and one being a retro orientation with no ordered adenosine moiety. Diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) bind with one adenine positioned at the B2 subsite, the polyphosphate chain extending across the P1 subsite in an ill-defined conformation, and a disordered second adenosine moiety. Diadenosine pentaphosphate (Ap5A), the most avid inhibitor of this series, binds in a completely ordered fashion with one adenine positioned conventionally at the B2 subsite, the polyphosphate chain occupying the P1 and putative P(-1) subsites, and the other adenine bound in a retro-like manner at the edge of the B1 subsite. The binding mode of each of these inhibitors has features seen in previously determined structures of adenosine diphosphates. We examine the structure-affinity relationships of these inhibitors and discuss the implications for the design of improved inhibitors.

Influence of naturally‐occurring 5′‐pyrophosphate‐linked substituents on the binding of adenylic inhibitors to ribonuclease a: An X‐ray crystallographic study

“…The manner in which this compound binds to EDN (PDB entry 2C01)24 differs from that observed here with RNase A. A mixture of two conformations is present, neither of which is particularly well ordered.…”

“…The inhibitor is disordered beyond the 5′-γ-phosphate. (b) Comparison with the EDN·Ap 3 A complex (PDB entry 2C02) 24. The two complexes were superposed on the basis of the C α positions of key nucleotide-binding residues (from RNase A, mol A: Q11, H12, K41, T45, H119, and F120; from EDN: Q14, H15, K38, T42, H129, and L130).…”

“…When bound to EDN (PDB entry 2C02),24 the conformation of the second adenosine moiety is likewise disordered, but a syn base conformation is not observed. The reason for this is the need to avoid an overlap between the O3′ edge of the ribose and the side chain of Trp7 (a residue whose counterpart in RNase A is Ala4) (Figure 4b).…”

“…X-ray crystallographic studies of complexes formed between phosphoadenosine-based inhibitors and RNase A,20–22 EDN,23,24 and ECP25 have shown that these compounds bind minimally to the P 1 and B 2 subsites but can also make additional interactions further afield depending on the nature of substitution. Exploration of the more peripheral interactions may lead to the development of inhibitors that are specific to particular ribonuclease homologs.…”

The1H,13C,15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

“…Although it is still difficult to speculate on the presence of additional substrate binding sites in the present ECP structures, a putative P 21 site has been proposed, from the ECP-2 0 5 0 ADP complex and from the comparison with RNase A and EDN complexes. 31,32 In the NMR structures, the Arg34 residue shows two alternate conformations, one of which could probably mimic the interaction found in the ECP-2 0 5 0 ADP complex between Arg34 and the 5 0 phosphate group. Regarding His15, there is an interesting parallel between the active sites of ECP and RNase 1 in mammals and those of the ribotoxin a-sarcin 33 and ribonucleases of the RNase T1 family in microbes.…”