FIGURE 9.

Phylogeny and structural conservation of C. glabrata EpaA domains. A, conservation and variability of individual residues of Epa family A domains. Shown is a structure-based sequence alignment of the 17 Epa paralogs from C. glabrata CBS138 as analyzed in this study. The alignment was generated using a local copy of the T-Coffee software implemented with 3DCoffee (43, 52) in combination with the three-dimensional structure of Epa1A (14) and was further processed using the ConSurf server (41, 53). The degree of conservation/variability of individual residues is color-coded according to the ConSurf-server, and yellow letters show minor reliability. Positions of the loops L1, L2, and L3, which form the outer binding pocket, and the calcium-binding loops CBL1 and CBL2, which constitute the inner binding pocket, are indicated above the sequences. Indicated below the sequences are positions I–IV of CBL2, the positions of the DcisD (DD) motif, and the Asn residue of CBL2 that form the DD-N structural motif, as well as positions of the Trp residue of loop L3 and the Arg residue of CBL2 (position I), which constitute the W-R corner of the binding pocket. Arrows indicate three further residues (70, 106, 122) discussed in the text. B, phylogenetic tree of EpaA domains. The tree was created with the MEGA6 software using 500 bootstrap replications (44) and is based on the structure-guided multiple sequence alignment described in A. Bootstrapping values above 70% are indicated. Functional classification obtained by glycan array analysis is indicated by white (class I), black (class II), or gray (class III) circles. Sequence motifs of CBL2 positions II–IV are shown in turquoise. A bar refers to phylogenetic distances. C, conservation of surface properties of EpaA domains. A structural model of Epa1A is shown on the right, which depicts conserved and variable surface residues. The degree of conservation/variability is color-coded and was obtained by using the ConSurf server (41, 53) and the multiple sequence alignment shown in A. The ligand binding pocket is presented on the left and shows highly conserved residues, including the DcisD motif of CBL1 and an asparagine of CBL2, which both confer coordination of the Ca²⁺ ion, as well as a tryptophan residue of loop L3 and an arginine residue at position I of CBL2, which form a specific corner of the inner binding pocket. In contrast, residues at positions II–IV of CBL2 are highly variable.