GQSEYFDRGTQ

1. X-ray crystallography (298 K; pH: 7; CTY30S form 1; MES (pH 6 - 7) 100 mM; PEG 2000 monomethyl ether (14 - 20%); Protein (PBS buffer))


2. X-ray crystallography (298 K; pH: 7; CTY30S form 2; D-galactose (Buffer G) 300 mM; PEG 3350 (20%); Protein (Buffer G); Sodium citrate 100 mM)


3. X-ray crystallography (298 K; pH: 7; CTY30S form 3; Kemptide 250 mM; MES (pH 6 - 7) 100 mM; PEG 2000 monomethyl ether (14 - 20%); Protein (PBS buffer))


4. X-ray crystallography (298 K; pH: 7; CT-wt form 1; D-galactose (Buffer G) 300 mM; Magnesium acetate 150 mM; PEG 3350 (18-20%); Protein (Buffer G))


5. X-ray crystallography (298 K; pH: 7; CT-wt form 2; D-galactose (Buffer G) 300 mM; Lithium citrate 200 mM; PEG 3350 (20%); Protein (Buffer G))

1. O'Neal CJ, Amaya EI, Jobling MG, Holmes RK, Hol WG. "Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism." Biochemistry. 2004; 43(13): 3772-82. PubMed: 15049684
   
   

After modification by limited proteolysis and/or disulfide bond reduction, this activation loop is thought to undergo a order-to-disorder transition that results in the destabilization of the active site loop.

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Of the three CTY30S crystal forms, 1 and 3 show this region to be fully disordered, whereas residues 48-55 are shown to be disordered in form 2, with high B factors being seen for residues 45 - 47. The loop is ordered in the wt-CT, however, form 2 indicated high B factors for some of the residues along the loop, indicating a propensity towards disorder.

TQTGFVRHDD

1. X-ray crystallography (298 K; pH: 7; CTY30S form 1; MES (pH 6 - 7) 100 mM; PEG 2000 monomethyl ether (14 - 20%); Protein (PBS buffer))


2. X-ray crystallography (298 K; pH: 7; CTY30S form 2; D-galactose (Buffer G) 300 mM; PEG 3350 (20%); Protein (Buffer G) 100 mM; Sodium citrate)


3. X-ray crystallography (298 K; pH: 7; CTY30S form 3; Kemptide 250 mM; MES (pH 6 - 7) 100 mM; PEG 2000 monomethyl ether (14 - 20%); Protein (PBS buffer))


4. X-ray crystallography (298 K; pH: 7; CT-wt form 1; D-galactose (Buffer G) 300 mM; Magnesium acetate 150 mM; PEG 3350 (18-20%); Protein (Buffer G))


5. X-ray crystallography (298 K; pH: 7; CT-wt form 2; D-galactose (Buffer G) 300 mM; Lithium citrate 200 mM; PEG 3350 (20%); Protein (Buffer G))

1. O'Neal CJ, Amaya EI, Jobling MG, Holmes RK, Hol WG. "Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism." Biochemistry. 2004; 43(13): 3772-82. PubMed: 15049684
   
   

It is thought that destabilization of the active site loop makes it more flexible, thus allowing substrates to enter the active site, which undergo ADP-ribosylation.

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The active site loop is ordered in the wild-type structures, in addition to CTY30S forms 1 and 2. With the addition of the kemptide inhibitor to CTY30S form 3, poor electron density was seen for residues 66 - 71, a possible indication of a order-to-disorder transition of the active site loop when substrate binds.

QSDIDTHNRIK

1. X-ray crystallography (298 K; pH: 7; CTY30S form 1; MES (pH 6 - 7) 100 mM; PEG 2000 monomethyl ether (14 - 20%); Protein (PBS buffer))


2. X-ray crystallography (298 K; pH: 7; CTY30S form 2; D-galactose (Buffer G) 300 mM; PEG 3350 (20%); Protein (Buffer G); Sodium citrate 100 mM)


3. X-ray crystallography (298 K; pH: 7; CTY30S form 3; Kemptide 250 mM; MES (pH 6 - 7) 100 mM; PEG 2000 monomethyl ether (14 - 20%); Protein (PBS buffer))


4. X-ray crystallography (298 K; pH: 7; CT-wt form 1; D-galactose (Buffer G) 300 mM; Magnesium acetate 150 mM; PEG 3350 (18-20%); Protein (Buffer G))


5. X-ray crystallography (298 K; pH: 7; CT-wt form 2; D-galactose (Buffer G) 300 mM; Lithium citrate 200 mM; PEG 3350 (20%); Protein (Buffer G))

1. O'Neal CJ, Amaya EI, Jobling MG, Holmes RK, Hol WG. "Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism." Biochemistry. 2004; 43(13): 3772-82. PubMed: 15049684
   
   

All crystal structures showed an elongated conformation for the disordered region of A2. This region extends through the B5 pore of of CT-subunit B in order to interact with the KDEL receptor.

1. Lockman HA, Galen JE, Kaper JB. "Vibrio cholerae enterotoxin genes: nucleotide sequence analysis of DNA encoding ADP-ribosyltransferase." J Bacteriol. 1984; 159(3): 1086-9. PubMed: 6090390
   
   
2. Lockman H, Kaper JB. "Nucleotide sequence analysis of the A2 and B subunits of Vibrio cholerae enterotoxin." J Biol Chem. 1983; 258(22): 13722-6. PubMed: 6315707
   
   

Crystallography by O'Neal et al (2004) was performed on the full length protein minus the signal peptide (aa 1-18), therefore the disordered regions are attached to the parent protein rather than cleavage products (subunits A1 and A2). DP regions are numbered according the the whole protein, the O'Neal paper disregards the signal peptide (author's aa 1 = DisProt/UniProt aa 19).