(1)H(N), (13)C, and (15)N resonance assignments of the CDTb-interacting domain (CDTaBID) from the Clostridium difficile binary toxin catalytic component (CDTa, residues 1-221)

Abstract

Once considered a relatively harmless bacterium, Clostridium difficile has become a major concern for healthcare facilities, now the most commonly reported hospital-acquired pathogen. C. difficile infection (CDI) is usually contracted when the normal gut microbiome is compromised by antibiotic therapy, allowing the opportunistic pathogen to grow and produce its toxins. The severity of infection ranges from watery diarrhea and abdominal cramping to pseudomembranous colitis, sepsis, or death. The past decade has seen a marked increase in the frequency and severity of CDI among industrialized nations owing directly to the emergence of a highly virulent C. difficile strain, NAP1. Along with the large Clostridial toxins expressed by non-epidemic strains, C. difficile NAP1 produces a binary toxin, C. difficile transferase (CDT). As the name suggests, CDT is a two-component toxin comprised of an ADP-ribosyltransferase (ART) component (CDTa) and a cell-binding/translocation component (CDTb) that function to destabilize the host cytoskeleton by covalent modification of actin monomers. Central to the mechanism of binary toxin-induced pathogenicity is the formation of CDTa/CDTb complexes at the cell surface. From the perspective of CDTa, this interaction is mediated by the N-terminal domain (residues 1-215) and is spatially and functionally independent of ART activity, which is located in the C-terminal domain (residues 216-420). Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of a 221 amino acid, ~26 kDa N-terminal CDTb-interacting domain (CDTaBID) construct by heteronuclear NMR spectroscopy. These NMR assignments represent the first component coordination domain for a family of Clostridium or Bacillus species harboring ART activity. Our assignments lay the foundation for detailed solution state characterization of structure-function relationships, toxin complex formation, and NMR-based drug discovery efforts.

Keywords: ADP-ribosyltransferase (ART); Binary toxin; CDTa; CDTb-interacting domain (BID); Clostridium difficile infection (CDI).

Figure 1

2D [¹H-¹⁵N]-TROSY-HSQC of the N-terminal CDTb-interaction domain of CDTa (1mBID) recorded on a Bruker Avance III 950 MHz spectrometer at pH 6.0 and 298 K. An enlarged view of the most crowded region of the spectrum is shown in the top-left corner. Backbone amide N-H correlations are labeled with the single-letter amino acid code and residue number of the mature native protein. Asterisks (*) mark the coordinates of residues undetectable at the displayed contour level (S75 and G189) and crosses (†) indicate aliased arginine sidechain correlations.

Figure 2

Characterization of the N-terminal CDTb-interaction domain of CDTa (1mBID) from Clostridium difficile based on NMR chemical shifts. (a) Raw chemical shift deviations of Cα and Cβ carbons (Δδ(Cα)-Δδ(Cβ)) with respect to corresponding random coil values are plotted against residue number. Positive and negative values indicate α-helix and β-strand character, respectively. Panel (b) shows the probability of secondary structure formation as predicted by Talos+, with α-helices represented by blue bars and β-strands by red bars. Amino acid stretches that meet the requirements for secondary structure definition are indicated by blue and red boxes as α1–4 and β1–5. Heteronuclear NOE relaxation parameters acquired at 950 MHz with a 5-s relaxation delay (closed black circles) indicate mobile regions of 1mBID that are consistent with Talos+ predictions.