Mechanisms for Intragenic Complementation at the Human Argininosuccinate Lyase Locus

Abstract

Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.

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

“…The construction of pET-3c vectors expressing wild-type human ASL, and the D87G and Q286R single point mutants with a Cterminal His 6 tag is described elsewhere (20). For the expression of ASL/␦2 crystallin hybrid tetramers the ASL wild-type or mutant plasmid was digested with Eco47III, and the resulting 1,915-bp fragment was ligated into the 4,818-bp fragment resulting from the digestion of the ␦2 crystallin wild-type or mutant plasmid with Bst1107I and Bsp68I.…”

“…Protein Expression and Purification-All proteins were overexpressed in E. coli strain BB101, genotype ara ⌬(lac proAB) ⌬slyD (kan r ) nalA argEam rif thi F'[lacI q proAB ϩ ] ( DE3) and purified by nickel affinity chromatography as described previously (20). Purification yielded ϳ30 mg of 95% pure protein/liter of cultured cells.…”

“…Extensive intragenic complementation is observed at the ASL locus in humans (19). By characterizing the effects of different pairs of mutations in ASL, two general mechanisms by which intragenic complementation can occur have been identified (20). Comple-* This work was supported in part by Operating Grant GP0155209 from the Natural Sciences and Engineering Research Council of Canada (to P. L. H. and A. R. D.).…”

“…The ASL subunit interface has been shown to be extremely stable (20). The tetramers do not dissociate at room temperature.…”

“…The tetramers do not dissociate at room temperature. Subunits will exchange extremely slowly at 0°C, reaching equilibrium only after several days (20). However, single amino acid substitutions of buried residues in the interface can vastly destabilize the protein.…”