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1.
Science ; 309(5737): 1093-6, 2005 Aug 12.
Article in English | MEDLINE | ID: mdl-16099990

ABSTRACT

The Vibrio cholerae bacterium causes devastating diarrhea when it infects the human intestine. The key event is adenosine diphosphate (ADP)-ribosylation of the human signaling protein GSalpha, catalyzed by the cholera toxin A1 subunit (CTA1). This reaction is allosterically activated by human ADP-ribosylation factors (ARFs), a family of essential and ubiquitous G proteins. Crystal structures of a CTA1:ARF6-GTP (guanosine triphosphate) complex reveal that binding of the human activator elicits dramatic changes in CTA1 loop regions that allow nicotinamide adenine dinucleotide (NAD+) to bind to the active site. The extensive toxin:ARF-GTP interface surface mimics ARF-GTP recognition of normal cellular protein partners, which suggests that the toxin has evolved to exploit promiscuous binding properties of ARFs.


Subject(s)
ADP-Ribosylation Factors/chemistry , ADP-Ribosylation Factors/metabolism , Cholera Toxin/chemistry , Cholera Toxin/metabolism , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/genetics , Amino Acid Sequence , Binding Sites , Cholera Toxin/genetics , Crystallography, X-Ray , Dimerization , Evolution, Molecular , Guanosine Diphosphate/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , NAD/metabolism , Protein Binding , Protein Conformation , Protein Structure, Secondary
2.
Int J Med Microbiol ; 294(4): 217-23, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15532979

ABSTRACT

Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels. Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated. At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.


Subject(s)
Bacterial Toxins/chemistry , Cholera Toxin/chemistry , Enterotoxins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Vibrio cholerae/metabolism , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/metabolism , Crystallography, X-Ray , Enterotoxins/antagonists & inhibitors , Enterotoxins/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/metabolism , Humans , Models, Molecular , Structure-Activity Relationship
3.
Biochemistry ; 43(13): 3772-82, 2004 Apr 06.
Article in English | MEDLINE | ID: mdl-15049684

ABSTRACT

Cholera toxin (CT) is a heterohexameric bacterial protein toxin belonging to a larger family of A/B ADP-ribosylating toxins. Each of these toxins undergoes limited proteolysis and/or disulfide bond reduction to form the enzymatically active toxic fragment. Nicking and reduction render both CT and the closely related heat-labile enterotoxin from Escherichia coli (LT) unstable in solution, thus far preventing a full structural understanding of the conformational changes resulting from toxin activation. We present the first structural glimpse of an active CT in structures from three crystal forms of a single-site A-subunit CT variant, Y30S, which requires no activational modifications for full activity. We also redetermined the structure of the wild-type, proenzyme CT from two crystal forms, both of which exhibit (i) better geometry and (ii) a different A2 "tail" conformation than the previously determined structure [Zhang et al. (1995) J. Mol. Biol. 251, 563-573]. Differences between wild-type CT and active CTY30S are observed in A-subunit loop regions that had been previously implicated in activation by analysis of the structure of an LT A-subunit R7K variant [van den Akker et al. (1995) Biochemistry 34, 10996-11004]. The 25-36 activation loop is disordered in CTY30S, while the 47-56 active site loop displays varying degrees of order in the three CTY30S structures, suggesting that disorder in the activation loop predisposes the active site loop to a greater degree of flexibility than that found in unactivated wild-type CT. On the basis of these six new views of the CT holotoxin, we propose a model for how the activational modifications experienced by wild-type CT are communicated to the active site.


Subject(s)
Cholera Toxin/chemistry , Cholera Toxin/genetics , Mutagenesis, Site-Directed , Bacterial Toxins/chemistry , Binding Sites/genetics , Crystallization , Crystallography, X-Ray , Enterotoxins/chemistry , Escherichia coli Proteins/chemistry , Galactose/chemistry , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Binding/genetics , Protein Conformation , Protein Structure, Secondary/genetics , Protein Subunits/chemistry , Protein Subunits/genetics , Serine/genetics , Structure-Activity Relationship , Tyrosine/genetics
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