Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from <i>Clostridium perfringens</i>

<div><p><i>Clostridium perfringens</i> is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the <i>Cp</i>GH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.</p></div

Oligonucleotide primers used for cloning of the <i>Cp</i>GH31 CBM32s.

<p>Oligonucleotide primers used for cloning of the <i>Cp</i>GH31 CBM32s.</p

Comparison of the GalNAc binding sites of the <i>Cp</i>GH31 CBM32s.

<p>The variable loop regions of (A) CBM32-1 (red), (B) CBM32-2 (orange), and (C) CBM32-3 (violet) contain a similar complement of residues involved in the recognition of GalNAc (green), including one or more aromatic residues. The structural conservation of key residues in the variable loop regions of CBM32-1 and CBM32-3 (His990, Phe1085, Tyr972 and His1671, Tyr1774, Tyr1674, respectively) allowed for GalNAc to be modeled into the binding site of CBM32-1. Specifically, <i>Cp</i>GH31 CBM32-3:GalNAc was identified as the top structural homologue of CBM32-1 using the DALI server (Z-score of 18.1; backbone r.s.m.d. of 1.9 Å), and the two structures were superimposed in order to position GalNAc into the binding site of CBM32-1. The binding site of CBM32-2 is unique as these residues are not conserved, but rather replaced by an extensive suite of residues involved in the coordination of GalNAc via hydrogen bonding. Residues are represented by their single-letter amino acid code. (D) Amino acid sequence alignment of the three <i>Cp</i>GH31 modules. Positions comprising conserved amino acid residues are identified by white single-letter code and highlighted in red while positions displaying amino acid residues of similar physicochemical properties are identified by red-single letter code. An asterisk denotes amino acid residues of <i>Cp</i>GH31 CBM32-1 implicated in GalNAc recognition by NMR titrations while those coordinating GalNAc in the <i>Cp</i>GH31 CBM32-2 and CBM32-3 are identified by pound and ampersand symbols, respectively. The sequence alignment was created using CLUSTAL OMEGA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref046" target="_blank">46</a>] and displayed using ESPript [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref047" target="_blank">47</a>].</p

Amino acid sequence comparison of GalNAc-binding <i>C</i>. <i>perfringens</i> CBM32s.

<p>Sequence alignment of the three <i>Cp</i>GH31 CBM32 modules with other functionally characterized CBM32 modules from the following family 33, family 84, and family 89 glycoside hydrolases with specificity for galacto- or gluco-configured sugars: <i>Cp</i>GH33 CBM32, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref021" target="_blank">21</a>]; <i>Cp</i>GH84A CBM32-1, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref024" target="_blank">24</a>]; <i>Cp</i>GH84A CBM32-2, GlcNAc specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref023" target="_blank">23</a>]; <i>Cp</i>GH84C CBM32, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref022" target="_blank">22</a>]; <i>Cp</i>GH89 CBM32-3 and CBM32-4, GlcNAc-α-1,4-Gal specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref020" target="_blank">20</a>]; <i>Cp</i>GH89 CBM32-5, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref020" target="_blank">20</a>]. Positions comprising conserved amino acid residues are identified by white single-letter code and highlighted in red while positions displaying amino acid residues of similar physicochemical properties are identified by red-single letter code. Sugar-coordinating amino acid residues in each CBM32 seequence are identified by black boxes. The sequence alignment was created using CLUSTAL OMEGA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref046" target="_blank">46</a>] and displayed using ESPript [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref047" target="_blank">47</a>].</p

GalNAc binding determinants of <i>Cp</i>GH31 CBM32-2.

<p>(A) Backbone cartoon representation of CBM32-2 (grey) in complex with GalNAc (green), solved to a resolution of 2.00 Å. The associated calcium ion is depicted as a blue sphere. <i>F</i>_<i>obs</i><i>-F</i>_<i>calc</i> electron density maps of GalNAc (green) bound to peptide chains A and B of the CBM32-2:GalNAc structure are shown in green mesh and contoured to 3.0 σ. (B) GalNAc (green) is bound to CBM32-2 by a several aromatic and polar residues (orange) via direct and water-mediated hydrogen bonds. Associated water molecules are shown as cyan spheres and hydrogen bonds are depicted by dashed lines. The stacking interaction is mediated by Trp1359. (C) The shallow GalNAc-specific binding site of CBM32-2 (shown in grey) accommodates the O4 hydroxyl group of the ligand in an axial position only. The sugar associates with the side chain of Trp1359 (light purple) and forms numerous hydrogen-bonding interactions (magenta) that target the O6 hydroxyl and 2-acetamido groups on either end of the sugar.</p

<i>Cp</i>GH31 CBM32-3 recognizes GalNAc with the same set of residues employed by canonical galactose-binding CBM32s.

<p>(A) Backbone cartoon structure overlay of the X-ray crystal structures of CBM32-3 in the apo-form (red) and in complex with galactose (grey) and GalNAc (orange), determined to 1.58 Å, 1.48 Å and 2.50 Å, respectively. Galactose is shown in green, and GalNAc in magenta. The Ca²⁺ ion observed in all three structures is depicted as a blue sphere. (B) Electron density of GalNAc bound to the single peptide chain of the CBM32-3:GalNAc structure, with the <i>F</i>_<i>obs</i><i>-F</i>_<i>calc</i> electron density map contoured to 2.5 σ. (C) Galactose bound to monomer chains A and C in the CBM32-3:galactose complex structure, with the <i>F</i>_<i>obs</i><i>-F</i>_<i>calc</i> electron density map contoured to 3.0 σ. (D) Expanded view of structural overlay of the ligand-coordinating residues from the galactose-bound (grey) and GalNAc-bound (orange) structures from (A). The same set of residues is involved in binding both ligands via hydrogen bonds (shown as black dashes). (E) A structural overlay of <i>Cp</i>GH31 CBM32-3 (grey) bound to galactose (green, coordinating residues denoted) with <i>Cp</i>GH84C CBM32 (magenta, coordinating residues underlined) bound to LacNAc (orange, PDB code 2J1E; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref022" target="_blank">22</a>]).</p

Carbohydrate binding preferences of the <i>Cp</i>GH31 CBM32 modules.

<p>Regions for the two-dimensional ¹H-¹⁵N HSQC spectra of 100 μM CBM32-1, 184 μM CBM32-2, and 100 μM CBM32-3 at pH 6.9 in the absence (black) and presence (red) of 8 mM galactose (Gal), glucose (Glc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc) or N-glucosamine (GlcN).</p

Identification of the <i>Cp</i>GH31 CBM32-1 GalNAc binding site.

<p>(A) Region of overlaid ¹H-¹⁵N HSQC spectra of 500 μM CBM32-1 with increased amounts of GalNAc. (B) Backbone cartoon representation of the X-ray crystal structure of apo-CBM32-1 with residues whose backbone amide resonances were significantly perturbed (>1 standard deviation above the mean chemical shift change) in the presence of GalNAc shown in red. The calcium ion is shown as a blue sphere. (C) Surface representation of apo-CBM32-1 on which those significantly perturbed backbone amide resonances are displayed in red and identified in white as single-letter amino acid code. (D) Amino acid residues of CBM32-1 (shown as sticks and identified by single-letter code and position in <i>Cp</i>GH31 sequence) whose backbone amide resonances display significant GalNAc-induced chemical shift changes are coloured red on a backbone cartoon representation of apo-CBM32-1. (E) Comparison of the GalNAc binding sites of <i>Cp</i>GH31 CBM32-1 (grey, select residues denoted) and <i>Cp</i>GH31 CBM32-3 (magenta, residues underlined) reveal structural conservation of Phe1985 and His990 with Tyr1774 and His1671, respectively. Tyr972 and Tyr1674 are located in different variable loop regions but are similarly positioned in the binding site.</p

Modular architecture of <i>Cp</i>GH31.

<p>This enzyme comprises an N-terminal family 31 glycoside hydrolase catalytic module (GH31; dark grey), two fibronectin-type III modules (FN3; black), three family 32 carbohydrate-binding modules (CBM32; white), a putative cohesin module (COH; grey), a bacterial immunoglobulin-like 2 module (BIG_2; grey) and a module of unknown function (UNK; grey). The amino acid borders of the CBM32s are indicated accordingly.</p