X-ray Structure of Recombinant Ricin A-Chain at 1.8 Å Resolution

Weston, Simon A.; Tucker, Alec D.; Thatcher, David R.; Derbyshire, D.J.; Pauptit, Richard A.

doi:10.1006/jmbi.1994.1739

Cited by 110 publications

(117 citation statements)

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“…ricin, abrin and modeccin toxin) or AB5 multimers (e.g. Shigella dysenteriae toxin and Shiga-like bacterial toxins) (Villafranca and Robertus, 1981;Montfort et al, 1987;Katzin et al, 1991;Robertus, 1991;Rutenber et al, 1991;Monzingo et al, 1993;Weston et al, 1994). The observed diversity in RIP architecture can be understood as a consequence of sequence fitness of protein structures constrained by thermodynamics and function.…”

Section: Resultsmentioning

confidence: 99%

Finding a New Vaccine in the Ricin Protein Fold

Olson¹,

Carra²,

Roxas-Duncan³

et al. 2004

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Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.

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Section: Resultsmentioning

confidence: 99%

Finding a New Vaccine in the Ricin Protein Fold

Olson¹,

Carra²,

Roxas-Duncan³

et al. 2004

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“…This bound form of the substrate positions the nucleophile for in-line attack on the scissile bond and is believed to be a prerequisite for cleavage (Yang et al 2001). Structural studies also suggest that in order for the active site of ricin to gain access to the scissile bond, the tetraloop unfolds (Weston et al 1994;Yang et al 2001); however, the structure of this unfolded form is unknown. It is also unclear if EF-G or EF-Tu binds to the canonical form or an altered form of the SRL RNA.…”

Section: Discussionmentioning

confidence: 99%

Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop structure at 1.4 Å resolution

Correll

Swinger²

2003

RNA

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GNRA tetraloops (N is A, C, G, or U; R is A or G) are basic building blocks of RNA structure that often interact with proteins or other RNA structural elements. Understanding sequence-dependent structural variation among different GNRA tetraloops is an important step toward elucidating the molecular basis of specific GNRA tetraloop recognition by proteins and RNAs. Details of the geometry and hydration of this motif have been based on high-resolution crystallographic structures of the GRRA subset of tetraloops; less is known about the GYRA subset (Y is C or U). We report here the structure of a GUAA tetraloop determined to 1.4 Å resolution to better define these details and any distinctive features of GYRA tetraloops. The tetraloop is part of a 27-nt structure that mimics the universal sarcin/ricin loop from Escherichia coli 23S ribosomal RNA in which a GUAA tetraloop replaces the conserved GAGA tetraloop. The adenosines of the GUAA tetraloop form an intermolecular contact that is a commonplace RNA tertiary interaction called an A-minor motif. This is the first structure to reveal in great detail the geometry and hydration of a GUAA tetraloop and an A-minor motif. Comparison of tetraloop structures shows a common backbone geometry for each of the eight possible tetraloop sequences and suggests a common hydration. After backbone atom superposition, equivalent bases from different tetraloops unexpectedly depart from coplanarity by as much as 48°. This variation displaces the functional groups of tetraloops implicated in protein and RNA binding, providing a recognition feature.

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“…3). The water molecule interacting with Arg-A170 closely approximates the position of N3 of adenine when bound to ricin (31). The oxygen atoms of formic acid occupy roughly the positions of N1 and N6 or N1 and N7 of the adenine in the two alternate orientations of the molecule of formic acid, and the carbon atom approximates the position of C6 of adenine.…”

Section: Stx2-mentioning

confidence: 97%

Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7

Fraser

Fujinaga

Cherney

et al. 2004

Journal of Biological Chemistry

266

280

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Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stx1. To investigate the differences among the Stxs, which are all AB 5 toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-Å resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit. Of the three types of receptor-binding sites in the B-pentamer, one has a different conformation in Stx2 than in Stx, and the carboxyl terminus of the A-subunit binds at another. Any of these structural differences might result in different mechanisms of action of the two toxins and the development of hemolytic uremic syndrome upon exposure to Stx2.

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X-ray Structure of Recombinant Ricin A-Chain at 1.8 Å Resolution

Cited by 110 publications

References 0 publications

Finding a New Vaccine in the Ricin Protein Fold

Finding a New Vaccine in the Ricin Protein Fold

Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop structure at 1.4 Å resolution

Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7

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