Passage
The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis. Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors. Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane. The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation. Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1) .
Figure 1 Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site. The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing. This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed. Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively. They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2) .
Figure 2 Percent cleavage of wild-type and mutant Tcd by autoprocessing
Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002.
-Which of the following amino acid residues is LEAST likely to be present in the Tcd central region?

Question

Passage
The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis. Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors. Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane. The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation. Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1) .

Figure 1 Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site. The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing. This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed. Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively. They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2) .

Figure 2 Percent cleavage of wild-type and mutant Tcd by autoprocessing
Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002.
-Which of the following amino acid residues is LEAST likely to be present in the Tcd central region?

Quizplus · Accepted Answer

11ecba2d_0ddd_d8bc_a3bf_1f7b72a37448_MD0008_00 The central region of TcdA and TcdB is a transmembrane domain that anchors the protein to the endosomal membrane. Transmembrane regions span cell membranes one or more times and often take on α-helical or transmembrane β-barrel arrangements. Membranes consist of phospholipid bilayers composed of inner and outer leaflets of phospholipids oriented with their hydrophilic head groups facing the aqueous environments and hydrophobic tails facing each other. Because any hydrophilic amino acid residues within the membrane would interact unfavorably with hydrophobic phospholipid tails, only membrane-spanning protein domains containing principally hydrophobic residues are thermodynamically stable. Histidine contains a basic, hydrophilic side chain. With a pK_a of ~6, a substantial percentage of histidine residues are protonated and positively charged at physiological pH, making them even more hydrophilic. Of the residues listed, histidine would be the least likely to be found within a transmembrane domain. (Choice A) The side chain of alanine is a hydrophobic methyl group and would interact well with phospholipid tails. (Choice C) The side chain of valine is a hydrophobic isopropyl group and is commonly found in transmembrane regions. (Choice D) The largely aliphatic side chain of methionine is relatively hydrophobic, and is frequently found in transmembrane regions. Educational objective: Transmembrane proteins contain hydrophobic transmembrane domains composed largely of nonpolar amino acids. They interact favorably with the hydrophobic tails of phospholipids in cellular membranes.

Passage The Bacterium Clostridium Difficile Secretes Protein Toxins TcdA and TcdB