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 -stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the -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 H2O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.is the catalytic subunit of ricin, a Centers for Disease Control and Prevention category B bioterrorism agent derived from Ricinus communis seeds. RTA catalyzes the depurination of an invariant adenosine residue, A 4234, within the GA 4234 GA tetraloop motif of the highly conserved sarcin-ricin loop of eukaryotic 28S rRNA (1). Clinical trials have exploited the toxicity of RTA in RTA-antibody constructs to kill leukemia and lymphoma cells (e.g., RTA conjugated to anti-CD22) (2-5). Side effects limit the utility of RTA immunotoxins (6, 7). Targeted inhibitors against ribosome-inactivating proteins (RIPs) could improve immunotoxin cancer therapies by rescuing normal cells following toxin treatment.Saporin-L1 (SAP), a homologue of RTA from Saponaria officinalis (soapwort) leaves, exhibits N-glycohydrolase activities on 80S ribosomes, poly(A) RNA, and other cellular . SAP releases multiple adenines from ribosomes, whereas RTA shows exquisite specificity.Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13-16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17, 18) (Fig. 1).Transition state analysis of RTA-mediated depurinations established that hydrolysis of adenine involves a ribocation intermediate, followed by attack of an activated water. Adenine activation is a major driving force for RTA catalysis (19,20). Efficient catalysis by RTA requires the invariant Glu-177 and Arg-180 residues (21-25). RTA and other RIPs have evolved to become near-perfect catalysts for mammalian ribosomes (21,(26)(27)(28). Here, we use transition state analogues to establish the catalytic site features contributing to this remarkable catalytic activity.RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17, 29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the...

Identification of a Novel Functional Domain of Ricin Responsible for Its Potent Toxicity

“…5). Previous studies have identified that Glu-177 is critical for the depurination activity of ricin (5,6,9,12). Taken together, these data suggested that the ␣-helix may modulate the enzymatic activity of ricin by controlling the side chain orientation of Glu-177.…”

“…Recently, the transition state analogues in structures of ricin and saporin ribosome-inactivating proteins were studied. The data confirmed that the invariant residues of RIPs in the catalytic active site of ricin were essential for the efficient catalysis by RTA (9). Although the biochemical properties of RIPs have been extensively studied, the enzymatic mechanism of RIPs is still elusive.…”

“…5B). Previous studies have demonstrated that there are four amino acids (Tyr-80, Tyr-123, Glu-177, and Arg-180) critical for the depurination activity of ricin (6,9,12,25). To investigate how the flexibility of the ␣-helix affects the enzyme activity of ricin, we performed a dihedral angle analysis.…”

Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

“…The epitope mainly con-tains the residues Asp 96 -Thr 116 , which forms the substrate binding pocket together with the residues Gly 120 -Asn 136 and residues Asp 201 -Ser 221 (7). To investigate whether 6C2 binding to RTA will change its structure near the enzyme activity center and eliminate the rRNA depurination activity of RTA, we compared the apo-form RTA (PDB code 2AAI) with the RTA in complex with 6C2 and a substrate analog (PDB codes 4KUC and 3HIO).…”

“…Recently, the structure of ricin was solved with transition state analogues bound, confirming that these invariant residues of the RIPs in the catalytic active site of ricin were essential for efficient catalysis by RTA (7). Although the biochemical properties of RIPs have been extensively studied, the enzymatic mechanism of RIPs remains elusive.…”