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Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

ABSTRACT: 8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.

SUBMITTER: Roday S

PROVIDER: S-EPMC2536774 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

Roday Setu S Saen-oon Suwipa S Schramm Vern L VL

Biochemistry 20070504 21

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (G ...[more]

PMID: 17477546

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Project description:The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, ?-lactamase (?lac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting ?15 and ?16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, ?-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (?lac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between ?15 and ?16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Dataset Information

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

Publications

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

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