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Vibrio cholerae produces a toxin of the A-B-type which causes a massive loss of liquid from the intestine. The holotoxin (choleragen) is a heterohexamer of the composition AB₅ (M_r 85620). The B-pentamer (choleragenoid) binds specificially to a glycolipid receptor (gangliosid) located on intestinal epithelial cells. The A-subunit is cleaved and the catalytically active aminoterminal part is transferred into the cell by an unknown mechanism. In the cell NAD is bound und used to ADP-ribosylate a GTP binding protein associated with the adenylate cyclase.

The frame to the left shows a wireframe model of the holotoxin (every chemical bond between non-hydrogen atoms is symbolized by a bar). To highlight structural elements the different protein chains are colored individually

Subunit A
Subunit B1
Subunit B2
Subunit B3
Subunit B4
Subunit B5

To simplify the schema the sidechains of the amino acids are deleted and a connecting line of the C_alpha-atoms is drawn instead

A-subunit
B-subunit

The A-subunit is synthesized as a protein of 240 amino acids and is cleaved by a bacterial protease between Arg₁₉₂ and Ser₁₉₄. In the protein crystal investigated here the chains A1 (1-192) and A2 (196-240) are visible. A disulfide bridge between Cys₁₈₇ and Cys₁₉₉ connects A1 and A2 kovalently. The reduction of this cystine is prerequisite to the liberation of the catalytically active part A1. In the complex fragment A2 is situated in a shallow groove of A1 . Within this groove (the surface of the protein is depicted for this part) there are numerous hydrophobic contacts toward A2 stabilizing the connection.

In protein A1 several units may be discerned. The first 132 amino acids constitute a globular domain containing two threestranded antiparallel pleated sheets and some short helical stretches . In this area there is the active center of the ADP ribosyle transferase . In this groove are situated Ser₆₁ and Glu₁₁₂ which take part in catalysis.

Another globular part holds the 31 carboxyterminal amino acids of A1 . This part of the molecule is strongly hydrophobic and contains six prolines .

The two globular parts are connected by a bridge of amino acids 133 to 161 . There is a hydrogen bridge between the backbone nitrogen of Asn₁ and the carbonyl oxygen of Leu₁₅₃ stabilizing the intrinsically flexible structure.

Protein chain A2 forms a continous helix which is bent at one point by 52° . This rigid chain fastenes the catalytic subunit to the B pentamer ; the bent part of the helix is in a central pore between the B subunits and is held in place by the surrounding helices . The last four amino acids (Lys Asp Glu Leu) at the carboxyl terminus of A2 correspond to the recognition sequence for the retention of proteins in the endoplasmatic reticulum (KDEL-motiv).

There are some hydrogen bridges between arginines in A1 (R143, R148) and threonine/glutamate in B (T78, E79) . Because of the rotation symmetric placement of the B subunits a rotational movement of the A subunit over B is possible.

The central helices of the B pentamer form a channel with a diameter of 11 Å facing the A subunit and 16 Å diameter on the opposite side binding to the membrane . Into this pore point the sidechains of amino acids with alternating positive and negative charge . By this interhelical salt bridges are possible without a net charge of the channel.

Besides a long helix directed towards the pore (and a short aminoterminal helix) the B subunits contain two threestranded sheets . The beta strands are oriented in a way to constitute pleated sheets with adjoining subunits:

For binding to the luminal side of intestinal epithelial cells the B pentamer recognizes the glycosidic motiv Gal-Nga-Gal-Glc-Sia:

Involved with the binding are the amino acids Glu₁₁, Tyr₁₂, His₁₃, Asn₁₄, Glu₅₁, Gln₅₆, His₅₇, Gln₆₁, Trp₈₈, Asn₉₀ and Lys₉₁ .

Literature:
R-G Zhang et al, The 2.4 Å crystal structure of cholera toxin B subunit pentamer: choleragenoid, J. Mol. Biol. 251 (1995) 550-562
R-G Zhang et al, The three-dimensional crystal structure of cholera toxin, J. Mol. Biol. 251 (1995) 563-573