Structural Studies of Duck δ1 and δ2 Crystallin Suggest Conformational Changes Occur during Catalysis^,

Abstract

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

“…With the exception of monomer B of the H171A protein for which residues 294-297 could not be modeled, the B-factors for the C3 loop of each H171A and H171N monomer are comparable to the average values for the rest of the protein (Table 1). Interestingly, the conformation of the C3 loop in the H171A-ADS and H171N-AMP•FUM structures resembles the conformation observed previously in the sulfate bound dδc1 protein 33 (Figure 3). …”

“…Domain 3 has been hypothesized to undergo a rigid body movement in ASL/ δ2-crystallin upon substrate binding and catalysis, with the effect of partially sequestering the substrate from solvent. 33 …”

“…This loop conformation has only been observed in the sulfate and phosphate bound forms of dδc1 and E. coli ASL (ec-ASL), respectively. 33,34 A closed C3 loop is thought to be the catalytically relevant conformation, as the highly conserved residues in this region are positioned to interact with the substrate. In the "open" conformation these residues are up to 8 Å away from the active site region.…”

“…Mutation of S295 and the equivalent S281 in dδc2 26 demonstrate the importance of this residue for enzymatic activity, but its exact role in catalysis has until now been unclear. S281 has been proposed to be the acid catalyst in ASL/ δ2-crystallin, 33 while in ec-ASL (S277) 34 and Bacillus sp. YM55-1 aspartase (S318) 10 it has been suggested to be involved in base catalysis.…”

Disruption of a Salt Bridge Dramatically Accelerates Subunit Exchange in Duck δ2 Crystallin

“…33% of the surface area of the monomer or 7,129 Å 2 is buried on tetramerization. The ASL and ␦2 crystallin monomer is composed of three structural domains (16,18). Five of the helices in the central domain of each monomer form a helical bundle.…”