Structural studies of duck delta2 crystallin mutants provide insight into the role of Thr161 and the 280s loop in catalysis

Abstract

Delta crystallin, a taxon-specific crystallin present in avian eye lenses, is homologous to the urea cycle enzyme ASL (argininosuccinate lyase). Although there are two delta crystallin isoforms in duck lenses, ddeltac1 (duck delta1 crystallin) and ddeltac2 (duck delta2 crystallin), only ddeltac2 is catalytically active. Previous structural studies have suggested that residues Ser283 and His162 in the multi-subunit active site of ddeltac2/ASL are the putative catalytic acid/base, while the highly conserved, positively charged Lys289 is thought to help stabilize the carbanion intermediate. The strict conservation of a small hydroxy-containing residue (Thr or Ser) at position 161 adjacent to the putative catalytic base, as well as its proximity to the substrate in the S283A ddeltac2 enzyme-substrate complex, prompted us to investigate further the role this residue. Structures of the active T161S and inactive T161D ddeltac2 mutants, as well as T161D complexed with argininosuccinate, have been determined to 2.0 A resolution. The structures suggest that a hydroxy group is required at position 161 to help correctly position the side chain of Lys289 and the fumarate moiety of the substrate. Threonine is probably favoured over serine, because the interaction of its methyl group with Leu206 would restrict its conformational flexibility. Residues larger than Thr or Ser interfere with substrate binding, supporting previous suggestions that correct positioning of the substrate's fumarate moiety is essential for catalysis to occur. The presence of the 280s loop (i.e. a loop formed by residues 270-290) in the 'open' conformation suggests that loop closure, thought to be essential for sequestration of the substrate, may be triggered by the formation of the carbanion or aci-carboxylate intermediates, whose charge distribution more closely mimics that of the sulphate ion found in the active-site region of the inactive ddeltac1. The 280s loop in ddeltac1 is in the closed conformation.

Figure 1. Sequence alignment showing the conserved residues in the δ crystallin, ASL, adenylosuccinate lyase, 3-carboxy-cis,cis-muconate lactonizing enzyme, fumarase and aspartase families against grey, orange, yellow, purple, blue and cyan backgrounds respectively

Abbreviations used: CRD1 and CRD2, δ1 and δ2 crystallin respectively; ARLY, ASL; PUR8, adenylosuccinate lyase; FUMC, fumarase C; ASPA, aspartase; PCAB, 3-carboxy-cis,cis-muconate lactonizing enzyme. The other letters represent the species names abbreviated according to the SwissProt nomenclature (e.g. ANAPL corresponds to Anas platyrhynchos, the domestic duck). The delimitation between the three structural domains is shown with arrows, while the location of the three conserved amino acid sequences in the ASL superfamily (C1, C2 and C3) is shown with boxes.

Figure 2. Stereo plot of the 2Fo-Fc σ_A weighted electron density for AS in the T161D–AS structure (contoured at 1σ)

The carbon atoms (light grey) corresponding to the fumarate moiety of the substrate have been labelled. Nitrogen and oxygen atoms are shown in medium and dark shades of grey respectively.

Figure 3. Structure of the T161D monomer and tetramer

(a) Schematic representation of the T161D–AS monomer (light grey). The side chain of Asp¹⁶¹ (medium grey) is shown in stick representation, with the three structural domains labelled. The AS substrate is rendered as a CPK model in dark grey. (b) The T161D–AS tetramer with each monomer shown in a different shade of grey. The tetramer exhibits D2 or 222 symmetry. The four active sites, formed at the interface of three different monomers, are circled, and the bound AS substrate (dark grey) is shown as depicted in (a).

Figure 4. Stereoview showing interactions between residue 161 and neighbouring Leu²⁰⁶ and Lys²⁸⁹ residues

Interactions are shown for the T161D–AS (coloured according to atom type), T161D (pink), T161S (blue), S283A–AS (green) and wild-type dδc2 (gold) and dδc1 (blue–grey) structures. The water molecules mediating the interaction between residue 161 and Lys²⁸⁹ are also shown as spheres. Ser¹⁶¹ is illustrated in a conformation that (a) prevents interaction with Lys²⁸⁹ or (b) allows interactions with Lys²⁸⁹. Due to the multi-subunit nature of each active site, the X_i notation represents residue X belonging to monomer i, where i=A, B, C or D.

Figure 5. Conformation of AS and its interaction with the protein

(a) Stereo view of AS as observed in the T161D–AS (coloured by atom type), S283A–AS (green) and H162N–AS (orange) structures. For comparison purposes, the sulphate molecule bound in the active-site region of dδc1 (pink) is also shown. (b)–(d) Schematic diagrams of the hydrogen-bonding network (dashed lines) and van der Waals interactions (solid lines) between the AS substrate, water molecules (grey-shaded boxes, denoted with a ‘W’) and active-site residues, as observed in the structures of (b) T161D–AS, (c) S283A–AS and (d) H162N–AS. Only amino acid residues that interact directly with the substrate whose distances are <3.2 Å have been drawn. (e) Stereo view of residues Asp¹⁶¹, His¹⁶², Glu²⁹⁶ and the substrate (AS) in the T161D–AS (coloured according to atom type), T161D (pink), T161S (light blue) and wild-type dδ2c (gold) structures. Due to the multi-subunit nature of each active site, the X_i notation represents residue X belonging to monomer i, where i=A, B, C or D.

Figure 6. Schematic representations of (a) the reaction mechanism of ASL/dδc2, (b) the reaction mechanism with the AS-nitro analogue and (c) the sulphate molecule

In (a), both the carbanion and aci-carboxylate forms are shown; in (b) both the carbanion and nitronate forms are shown. The charged groups discussed in the text are circled with dashed lines.