Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism.
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.
O'Neal
Claire J
CJ
Department of Chemistry and Biomolecular Structure Center, University of Washington, Seattle, Washington 98195, USA.
Amaya
Edward I
EI
Jobling
Michael G
MG
Holmes
Randall K
RK
Hol
Wim G J
WG
D001427:Bacterial Toxins / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004768:Enterotoxins / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D014443:Tyrosine; D012694:Serine; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli; D005690:Galactose