Crystal structure of an inactive duck delta II crystallin mutant with bound argininosuccinate

delta-Crystallin, the major soluble protein component of avian and reptilia n eye lenses, is highly homologous to the urea cycle enzyme, argininosuccin ate lyase (ASL). In duck lenses, there are two highly homologous delta crys tallins, delta I and delta II, that are 94% identical in amino acid sequenc e. While delta II crystallin has been shown to exhibit ASL activity in vitr o, delta I is enzymatically inactive. The X-ray structure of a His to Asn m utant of duck delta II. crystallin (H162N) with bound argininosuccinate has been determined to 2.3 Angstrom resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft formed by three different monomers in the tetramer. The active site of the H162N mutant structure reveals that the side chain of Glu 296 h as a different orientation relative to the homologous residue in the H91N m utant structure [Abu-Abed et al. (1997) Biochemistry 36, 14012-14022]. This shift results in the loss of the hydrogen bond between His 162 and Glu 296 seen in the H91N and turkey delta I crystallin structures; this H-bond is believed to be crucial for the catalytic mechanism of ASL/delta II crystall in. Argininosuccinate was found to be bound to residues in each of the thre e monomers that form the active site. The fumarate moiety is oriented towar d active site residues His 162 and Glu 296 and other residues that are part of two of the three highly conserved regions of amino acid sequence in the superfamily, while the arginine moiety of the substrate is oriented toward residues which belong to either domain 1 or domain 2. The analysis of the structure reveals that significant conformational changes occur on substrat e binding. The comparison of this structure with the inactive turkey delta I crystallin reveals that the conformation of domain 1 is crucial for subst rate affinity and that the delta I protein is almost certainly inactive bec ause it can no longer bind the substrate.