Portrait of an enzyme, a complete structural analysis of a multimodular {beta}-N-acetylglucosaminidase from Clostridium perfringens

Abstract

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens micro-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.

FIGURE 1.

Schematic of the modular structure of GH84C. At the N terminus is a Gram-positive secretion signal peptide (indicated by SP). This is followed by the family 84 catalytic module, a family 32 carbohydrate binding module (CBM), a cohesin module (Coh; previously called X82 module). At the C terminus of the enzyme is a fibronectin type III module (FN3). The amino acid numbers denoting the module boundaries are indicated.

FIGURE 2.

Structures of GH84C catalytic module and GH84C-CBM32 as determined using x-ray crystallography and SAXS. A and B show the crystal structures of GH84C catalytic module and GH84C-CBM32, respectively, in a ribbon representation. The arrow in B shows the C terminus of the CBM. C shows the GASBOR-generated SAXS envelope of GH84C-CBM32, whereas D shows the modules of GH84C-CBM32 manually fit into the SAXS envelope. E shows the model in D without the SAXS form. F shows the unmodified x-ray crystal structure, shown in B, fit into the SAXS-generated envelope. All of the structures are shown from identical orientations. The N-terminal domain is pictured in light blue, the catalytic TIM barrel is in orange, the helical bundle is in pale green, and the CBM in red.

FIGURE 3.

Structure of CBM32-Coh and CBM32-Coh·FIVAR-Doc and GH84C-CBM32-Coh as determined using SAXS. A shows, from left to right, the GASBOR-generated SAXS envelope for CBM32-Coh, the SAXS form with the structures of the CBM32 (red) and Coh (blue) modules fit into it, and the ribbon representation of the SAXS-derived model without the SAXS envelope. B shows the same for the CBM32-Coh module in complex with FIVAR-Doc from the μ-toxin (green denotes the FIVAR, pink the Doc, and yellow spheres the calcium atoms). C shows the crystal structure of GH84C-CBM32 that was overlapped with the GASBOR-generated SAXS envelope and model of CBM32-Coh, and D shows the crystal structure of GH84C-CBM32 that was overlapped with the GASBOR-generated SAXS envelope and model of the CBM32-Coh·FIVARDoc complex. E shows, from left to right, the GASBOR-generated SAXS envelope for GH84C-CBM32-Coh, the SAXS form with the structures of the catalytic module (purple, orange, and green), the CBM (red) and Coh (blue) modules fit into it, and the ribbon representation of the SAXS-derived model without the SAXS envelope.

FIGURE 4.

Structural features of the Coh-FN3 modular pair. A shows a ribbon representation of the 1.8-Å crystal structure of Coh-FN3. The Coh module is depicted in blue, and FN3 is shown in black. B shows the surface representation of Coh-FN3 colored according to electrostatic potential (red is negative, and blue is positive). The basic patch of FN3 is circled and expanded to show a patch of basic residues.

FIGURE 5.

Composite structure of GH84C. The overall structure of GH84C in complex with the FIVAR-Doc module from the μ-toxin determined by the amalgamation of the SAXS data and crystallographic data is shown. Two Asp residues, 297, proposed to be involved in stabilizing the conformation of the acetamido group of the N-acetylglucosamine, and 298, proposed to be the catalytic acid, of GH84C are shown as sticks interacting with the competitive inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenyl carbamate, which was modeled on the basis of the C. perfringens GH84C complex determined by Rao et al. (19). The CBM32 module is shown interacting with its carbohydrate ligand, N-acetyllactosamine, which was modeled on the basis of the previously determined complex of the CBM with this sugar (15). Arrows near the FN3 module represents possible motion of this module due to the flexible linker region. The structure is also shown rotated by 90° around the horizontal axis running parallel to the page (right). The same structure is shown below in a solvent-accessible surface representation. The N-terminal domain is pictured in light blue, the catalytic TIM barrel is colored orange, the helical bundle is in pale green, the CBM32 is red, the cohesin module is blue, and the FN3 module is black. The FIVAR and Doc modules from the μ-toxin are shown in green and pink, respectively. Calcium atoms are shown in yellow.