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>].