Recognition and interaction of small rings with the ricin A-chain binding site

Abstract

Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings.

Structure-Based Design and Characterization of Novel Platforms for Ricin and Shiga Toxin Inhibition

“…We carried out a systematic survey of compounds with differing patterns of hydrogen bond donors and acceptors to try to maximize these interactions. Computer modeling 23 suggested that 3-aminopyridothiadiazine would preserve many of the specific interactions observed for pterins but would juxtapose a partially negative sulfone with Arg 180 of RTA. Despite the suggestions of modeling, this compound, in fact, was not an inhibitor of the enzyme (Table 1).…”

“…We had predicted from energy calculations that guanine, rotated roughly 180°around the long of the purine base (a line from C8 to the midpoint of the N1-C2 bond) with respect to the adenine orientation, could bind and inhibit RTA. 23 As shown in Table 1, guanine has an IC 50 of 0.9 mM. We also tested xanthine as an inhibitor.…”

“…Energy calculations were used to investigate likely binding modes of other potential inhibitors, including tautomers of pterins and guanine. 23 These calculations suggested that guanine-like compounds could interact strongly with the RTA specificity pocket, and this served as a basis for the design and synthesis of a series of inhibitors described in this work. Figure 1 shows a cartoon representation of the interactions made between the pterin moiety of pteroic acid and the specificity pocket of RTA.…”

Change in Conformation with Reduction of α-Helix Content Causes Loss of Neutrophil Binding Activity in Fully Cytotoxic Shiga Toxin 1

“…These amino acids should have conserved the reciprocal spatial relationship required to fit the adenine residue during the preserved catalytic activity of the unfolded toxin. However, as shown in the crystallographic studies of ricin-or Stx-ligand complexes (41)(42)(43), few amino acids interact directly with adenine forming hydrogen bonds (Val-78, Ser-112, Tyr-114, and Arg-170; numbers as above). Thus, the unfolding of the ␣-helices containing the latter two amino acids (residues 114 -121 and 152-170) would probably interfere with the enzymatic activity of the toxin.…”

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

“…The conserved ␤-strands ␤5 and ␤6 form the base of the cleft, whereas the loops between ␤3 and ␤4, ␤8 and ␣2, and ␤7 and ␤8 form the walls. Four conserved residues, Arg 51 , Asp 70 , Thr 72 , and Asn 73 , represent the binding residues. The ribosome recognition site is a shallow cleft present on the surface, whereas the N-glycosidase activity site corresponds to a well defined deep pocket.…”