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 Figure 2, residues H757 and H759 of wild-type TcdA and TcdB, respectively, may function to:

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 Figure 2, residues H757 and H759 of wild-type TcdA and TcdB, respectively, may function to:

Quizplus · Accepted Answer

11ecba2d_0de3_08df_a3bf_0916a628457e_MD0008_00 The passage states that in wild-type Tcd proteins, IP6 binding induces a conformational change that moves the flap region away from the active site. This change is an example of allostery, a phenomenon in which the binding of an allosteric effector (eg, IP6) at an allosteric site (eg, the binding pocket) alters the activity at the catalytic site. Allosteric regulation occurs due to conformational changes induced in the protein upon binding of the effector and can include changes in the conformation of the active site itself, obstruction of the active site or, as in this case, exposure of the active site when the flap region moves. Unlike wild-type Tcd, autoprocessing occurs in H757A and H759A mutants even in the absence of IP6. This behavior supports the conclusion that H757 and H759 mediate an interaction necessary for the flap region to obstruct the active site in the absence of IP6. Conversion of histidine to alanine abolishes this interaction, so the flap region cannot fully block the active site in mutant proteins, resulting in a loss of allosteric control. (Choice A) The H → A mutations studied here result in some autoprocessing activity even when IP6 is absent. If these histidine residues were required for interaction with IP6, substituting them would decrease activity even at high IP6 concentrations. (Choice B) If the substituted histidines were acting as bases in the catalytic mechanism, mutating them to alanine would downregulate autoprocessing. Instead, the mutation results in increased activity. (Choice D) The H757A and H759A mutations involve replacing polar, charged histidine with neutral, nonpolar alanine. If histidine were opposing flap-active site interaction via charge repulsion, replacing it with an uncharged amino acid would reduce repulsion and facilitate interaction, decreasing enzymatic activity. Educational objective: Allosteric regulation occurs when an allosteric effector binds to the allosteric site and alters catalytic activity in the active site. Mutations that result in the instability of normal conformations can disrupt allosteric regulation.

Passage The Bacterium Clostridium Difficile Secretes Protein Toxins TcdA and TcdB