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.
-Based on information given in the passage, stabilizing interactions are most likely to occur between phosphate groups on IP6 and which of the following amino acids lining the Tcd binding pocket?

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.
-Based on information given in the passage, stabilizing interactions are most likely to occur between phosphate groups on IP6 and which of the following amino acids lining the Tcd binding pocket?

Quizplus · Accepted Answer

11ecba2d_0de0_beed_a3bf_4389959f5896_MD0008_00 The passage states that IP6 is stabilized in the binding pocket through salt bridging. In protein chemistry, salt bridging describes an electrostatic interaction that occurs between charged residues and ligands, stabilizing the protein conformation or strengthening protein-ligand interactions. Electrostatic interactions combine elements of both hydrogen bonding and ionic bonding. In proteins, it most commonly occurs between the anionic carboxylate of aspartic or glutamic acids and the cationic ε-amino group of lysine or the guanidinium group of arginine, but can potentially involve any pair of oppositely charged chemical groups. IP6 contains six phosphate groups, each bearing a −2 charge at physiological pH, so IP6 can only provide the anionic component of salt bridges; therefore, it must form salt bridges with cationic amino acids. Of the amino acids given, IP6 could potentially form a salt bridge with a cationic lysine residue. (Choice B) Glycine is neutral and nonpolar, and therefore cannot participate in salt bridge formation. Furthermore, salt bridging is not a hydrophobic interaction. (Choice C) Although cysteine residues can form covalent disulfide bonds with other cysteine residues, they do not participate in salt bridge formation. Salt bridging is a noncovalent interaction. (Choice D) Aspartate residues can participate in ionic interactions such as salt bridges, but are negatively charged, and IP6 can form a salt bridge only with a positively charged residue. Educational objective: Salt bridges are electrostatic attractions that contribute to the stability of folded proteins and interactions between proteins and ligands or substrates. Salt bridging combines elements of hydrogen bonding and ionic bonding, and occurs between oppositely charged moieties.

Passage The Bacterium Clostridium Difficile Secretes Protein Toxins TcdA and TcdB