Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Abstract

Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple pi-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the pi-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H(2)O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.

Fig. 1.

The structure of cyclic G(9-DA)GA 2′-OMe. 9-DA is a transition state mimic of 2′-deoxyadenosine. Atomic numbering for 9-DA follows that for purine nucleosides.

Fig. 2.

Protein folds of RTA and SAP. (A) SAP structure is depicted in a ribbon diagram. The N-terminus is blue, with a color change to green (residues 1–119), yellow, and red, progressing to the C-terminus (residues 120–257). The position of residue 119 is indicated. The interface between N-terminal and C-terminal domains is highlighted in the black box, and the hydrophobic side chains in the interface region are shown. (B) Stereo-view of the overlaid Cα traces of RTA (gray) and SAP (yellow) is shown. The side chains are shown for Ala-79, Asn-113, and Glu-121 of SAP (blue labels) and Ala-85, Asp-96, Asp-100, and Asn-113 of RTA (black labels). Residues 79–113 of SAP and residues 85–113 of RTA are shown in red and green, respectively, to highlight a region that differs. Both structures are from inhibitor-bound complexes (3HIW and 3HIO).

Fig. 3.

Catalytic site geometries for cyclic inhibitor G(9-DA)GA 2′-OMe bound to RTA and SAP. (A) π-Stacking interaction between the inhibitor and RTA is shown. The 2mFo-DFc electron density map and the inhibitor- and water nucleophile-omit mFo-DFc map were drawn in gray at a contour level of 1σ and in green at a contour level of 3σ, respectively. Glu-177 and Arg-180 are shown. (B) π-Stacking interaction between the inhibitor and SAP is shown. The 2mFo-DFc electron density map and the inhibitor- and water nucleophile-omit mFo-DFc map were drawn in gray at a contour level of 1σ and in green at a contour level of 3σ, respectively. Glu-174 and Arg-177 are indicated. (C) Comparison of inhibitor-bound RTA (Left, gray) and inhibitor-bound SAP (Right, yellow). The tyrosine involved in π-stacking is indicated. Distances in A and B are in angstroms.

Fig. 4.

Two-dimensional map of the cyclic transition state analogue inhibitor bound to the active sites of RTA (A) and SAP (B). The purines and residues involved in π-stacking are highlighted in orange. Water molecules are drawn as red dots. The hydrogen bonds are depicted as green dashed lines. The interaction between phosphodiester groups and the RIPs is shown in Figs. S2 and S3. The hydrogen bonds are depicted in green dashed lines (Å).

Fig. 5.

Stereo-view of the superimposed inhibitors bound to SAP (yellow, PDB ID code 3HIW) and an unbound GAGA tetraloop (green, PDB ID code 1Q9A). The partial nucleic acid backbone of the inhibitor (yellow) and GAGA tetraloop (green) is shown. Tyr-73 and 9-DA and the third guanosine are depicted in yellow. The last 3 nucleotides of the unbound GAGA tetraloop are shown in green. The 5′- and 3′-phosphate diesters of 9-DA are highlighted in the red circles.

Fig. 6.

Cyclic inhibitor in the active sites of RTA and SAP. (A) Surface of RTA is shown relative to the active site. Tyr-80, Asp-96, 9-DA, and the first guanosine of the inhibitor are shown. Negative, positive, and neutral charges are shown in red, blue, and gray, respectively. (B) Surface of SAP is drawn to illustrate the active site. Tyr-73, Glu-121, 9-DA, and the third guanosine of the inhibitor are shown. The negative, positive, and neutral charges are drawn in red, blue, and yellow, respectively.