Structural basis of antigen recognition: crystal structure of duck egg lysozyme

Abstract

Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, no structures of DEL have so far been reported, and the situation had been complicated by the presence of multiple isoforms and conflicting reports of primary sequence. Here, the structures of two DEL isoforms from the eggs of the commonly used Pekin duck (Anas platyrhynchos) are reported. Using structural analyses in combination with mass spectrometry, non-ambiguous DEL primary sequences are reported. Furthermore, the structures and sequences determined here enable rationalization of the binding affinity of DEL for well documented landmark anti-lysozyme antibodies.

Keywords: antigen recognition; duck egg lysozyme; glycosyl hydrolase; hen egg lysozyme.

Figure 1

Elution profile of Pekin DEL fractions from CM ion-exchange resin (top) in response to a linear salt gradient (50–450 mM NaCl, left to right). Also shown are SDS–PAGE examinations of certain fractions (bottom).

Figure 2

Alignment of the primary sequence of HEL (black text) with Pekin DEL sequences found in the literature (blue text; ‘DEL_Huang’ and ‘DEL_Kondo_’), with those structurally and/or confirmed by mass spectrometry described here (shaded blue; DEL-I, DEL-II ad DEL-III) as well as with sequences available for Khaki duck (red text), Egyptian goose and American wood duck (both grey text). Amino-acid identities are identical to HEL (indicated by a dash) unless explicitly stated. Ambiguities at positions 66 and 103 are highlighted in yellow, whilst arrows indicate the additional arginines in DEL sequences at positions 71, 79 and 100 (highlighted in green). Although derived by different means, the sequences of DEL_Huang and DEL_Kondo_DL-2 (marked with asterisks) effectively describe the same species (DEL-II).

Figure 3

Summary of mass-spectrometric analyses to support the proposed sequences for DEL-I, DEL-II and DEL-III. Sequences in blue text match the predicted endopeptidase Lys-C peptide masses, while underlined sequences match the predicted tryptic peptide masses. Sequences highlighted in pale yellow were matched to an MSMS spectrum for that sequence.

Figure 4

Composite OMIT 2F _o − F _c electron density (green mesh) contoured at 1σ for amino-acid positions 71, 79 and 100 for Pekin duck DEL-I and for cubic (DEL-IIIc) and orthorhombic (DEL-IIIo) crystal forms of DEL-III.

Figure 5

DEL-III cubic space group special position occupied by a phosphate anion which contacts Arg97 and Arg100. Composite OMIT 2F _o − F _c electron density (green mesh) contoured at 1σ as viewed down the crystallo­graphic threefold special position axis. Pairs of arginines projected from different lysozyme molecules are coloured green, grey and orange.

Figure 6

Structural fold dendogram of 24 structures encompassing the lysozyme family output by the DALI structural homology server (Holm & Laakso, 2016 ▸) and displayed using TreeDyn (Chevenet et al., 2006 ▸). The x axis pertains to structural similarity in units of DALI Z-score. Percentage amino-acid identities relative to DEL-I, DEL-III and HEL are shown in the right-hand columns, respectively.

Figure 7

Duck lysozymes superposed on the chicken enzyme. (a, b) Cartoons of Pekin DEL-I (orange) and two crystal forms of DEL-III (both light orange) superposed on HEL (PDB entry 1iee, grey). Amino-acid identities different from the chicken enzyme are shown as sticks. The arrow indicates the active-site cleft. (c–h) Ensemble superposed with the HEL–HyHEL5 complex (PDB entry 1yqv, cyan cartoon and surface) and the HEL–HyHEL10 complex (PDB entry 3d9a, slate cartoon and surface). (e) Position 68 is directly adjacent to HyHEL5, whilst positions 93 and 100 are directly adjacent to HyHEL10. (f) Substitution of Lys49 of the HyHEL10 light chain by threonine enhances binding fivefold to DELs which contain N97R. (g) The salt bridge found in the HEL–HyHEL5 complex is not supported by the R68K substitution in DELs. (h) The S100R substitution found in DEL-III disrupts binding to HyHEL10 owing to a steric clash with the HyHEL10 surface (shaded).