Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.

Type IVa pili are bacterial nanomachines required for colonization of surfaces, but little is known about the organization of proteins in this system. The Pseudomonas aeruginosa pilMNOPQ operon encodes five key members of the transenvelope complex facilitating pilus function. While PilQ forms the outer membrane secretin pore, the functions of the inner membrane-associated proteins PilM/N/O/P are less well defined. Structural characterization of a stable C-terminal fragment of PilP (PilP(Δ71)) by NMR revealed a modified ß-sandwich fold, similar to that of Neisseria meningitidis PilP, although complementation experiments showed that the two proteins are not interchangeable likely due to divergent surface properties. PilP is an inner membrane putative lipoprotein, but mutagenesis of the putative lipobox had no effect on the localization and function of PilP. A larger fragment, PilP(Δ18-6His), co-purified with a PilN(Δ44)/PilO(Δ51) heterodimer as a stable complex that eluted from a size exclusion chromatography column as a single peak with a molecular weight equivalent to two heterotrimers with 1:1:1 stoichiometry. Although PilO forms both homodimers and PilN-PilO heterodimers, PilP(Δ18-6His) did not interact stably with PilO(Δ51) alone. Together these data demonstrate that PilN/PilO/PilP interact directly to form a stable heterotrimeric complex, explaining the dispensability of PilP's lipid anchor for localization and function.

Pseudomonas aeruginosa Uses c-di-GMP Phosphodiesterases RmcA and MorA To Regulate Biofilm Maintenance.

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to identify rmcA and morA, two genes which encode bis-(3',5')-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the ΔrmcA and ΔmorA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms.IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process.

Conserved, unstructured regions in Pseudomonas aeruginosa PilO are important for type IVa pilus function.

Pseudomonas aeruginosa uses long, thin fibres called type IV pili (T4P) for adherence to surfaces, biofilm formation, and twitching motility. A conserved subcomplex of PilMNOP is required for extension and retraction of T4P. To better understand its function, we attempted to co-crystallize the soluble periplasmic portions of PilNOP, using reductive surface methylation to promote crystal formation. Only PilOΔ109 crystallized; its structure was determined to 1.7 Å resolution using molecular replacement. This new structure revealed two novel features: a shorter N-terminal α1-helix followed by a longer unstructured loop, and a discontinuous ß-strand in the second αßß motif, mirroring that in the first motif. PISA analysis identified a potential dimer interface with striking similarity to that of the PilO homolog EpsM from the Vibrio cholerae type II secretion system. We identified highly conserved residues within predicted unstructured regions in PilO proteins from various Pseudomonads and performed site-directed mutagenesis to assess their role in T4P function. R169D and I170A substitutions decreased surface piliation and twitching motility without disrupting PilO homodimer formation. These residues could form important protein-protein interactions with PilN or PilP. This work furthers our understanding of residues critical for T4aP function.

The 1.6 A crystal structure of E. coli argininosuccinate synthetase suggests a conformational change during catalysis.

BACKGROUND: Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. RESULTS: The crystal structure of E. coli AS (EAS) has been determined by the use of selenomethionine incorporation and MAD phasing. The structure has been refined at 1.6 A resolution in the absence of its substrates and at 2.0 A in the presence of aspartate and citrulline (EAS*CIT+ASP). Each monomer of this tetrameric protein has two structural domains: a nucleotide binding domain similar to that of the "N-type" ATP pyrophosphatase class of enzymes, and a novel catalytic/multimerization domain. The EAS*CIT+ASP structure clearly describes the binding of citrulline at the cleft between the two domains and of aspartate to a loop of the nucleotide binding domain, whereas homology modeling with the N-type ATP pyrophosphatases has provided the location of ATP binding. CONCLUSIONS: The first three-dimensional structures of AS are reported. The fold of the nucleotide binding domain confirms AS as the fourth structurally defined member of the N-type ATP pyrophosphatases. The structures identify catalytically important residues and suggest the requirement for a conformational change during the catalytic cycle. Sequence similarity between the bacterial and human enzymes has been used for providing insight into the structural and functional effects of observed clinical mutations.

Structure of E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases.

BACKGROUND: 5'-methylthioadenosine/S-adenosyl-homocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5'-methylthioribose and S-ribosylhomocysteine, respectively. While this enzyme is crucial for the metabolism of AdoHcy and MTA nucleosides in many prokaryotic and lower eukaryotic organisms, it is absent in mammalian cells. This metabolic difference represents an exploitable target for rational drug design. RESULTS: The crystal structure of E. coli MTA/AdoHcy nucleosidase was determined at 1.90 A resolution with the multiwavelength anomalous diffraction (MAD) technique. Each monomer of the MTA/AdoHcy nucleosidase dimer consists of a mixed alpha/beta domain with a nine-stranded mixed beta sheet, flanked by six alpha helices and a small 3(10) helix. Intersubunit contacts between the two monomers present in the asymmetric unit are mediated primarily by helix-helix and helix-loop hydrophobic interactions. The unexpected presence of an adenine molecule in the active site of the enzyme has allowed the identification of both substrate binding and potential catalytic amino acid residues. CONCLUSIONS: Although the sequence of E. coli MTA/AdoHcy nucleosidase has almost no identity with any known enzyme, its tertiary structure is similar to both the mammalian (trimeric) and prokaryotic (hexameric) purine nucleoside phosphorylases. The structure provides evidence that this protein is functional as a dimer and that the dual specificity for MTA and AdoHcy results from the truncation of a helix. The structure of MTA/AdoHcy nucleosidase is the first structure of a prokaryotic nucleoside N-ribohydrolase specific for 6-aminopurines.

Crystallization and preliminary X-ray analysis of a mutant duck delta II crystallin.

A duck delta II crystallin mutant, where histidine 178 has been replaced by an aspartic acid residue, has been purified from a bacterial expression system and subsequently crystallized. The crystals grow as flat plates, with unit cell dimensions a = 94.1 A, b = 99.9 A, c = 108.7 A and beta = 102 degrees. The crystals exhibit the symmetry of space group P2(1) and diffract to a minimum d-spacing of 2.8 A resolution. On the basis of density calculation four monomers (one tetramer) are estimated to be present in the asymmetric unit (Vm = 2.5 A3/Da). Self-rotation functions clearly show the presence of 222 non-crystallographic symmetry.

Expression, purification, crystallization and preliminary X-ray analysis of human argininosuccinic acid lyase.

Human argininosuccinic acid lyase (ASAL) has been expressed, purified and crystallized in several distinct crystal morphologies. At present only one form is suitable for X-ray diffraction analysis. These crystals grow as hexagonal prisms, with unit cell dimensions a = b = 104.6 A, c = 185.3 A and alpha = beta = 90 degrees, gamma = 120 degrees. The crystals exhibit the symmetry of space group P3(1)21 or its enantiomorph, P3(2)21 (indistinguishable crystallographically) and diffract to a minimum d-spacing of approximately 3.5 A.

Structures of partridge egg-white lysozyme with and without tri-N-acetylchitotriose inhibitor at 1.9 A resolution.

The three-dimensional structures of native partridge egg-white lysozyme (PEWL) and PEWL complexed with tri-N-acetylchitotriose inhibitor have been determined crystallographically and refined at 1.9 A resolution. Crystals of native and complexed protein are isomorphous and have space group and cell dimensions that are identical to those of hen egg-white lysozyme (HEWL) under similar crystallization conditions. Full occupancy of the trisaccharide in the inhibitor complex has allowed definitive modeling and refinement of all three sugar residues, located at subsites A, B, and C in the PEWL active site. A comparison has been made with HEWL/inhibitor complexes in which coordinates were either not refined (Blake CCF, et al., 1967, Proc R Soc B 167:378-388) or were refined at partial occupancy (Cheetham JC, Artymiuk PJ, Phillips DC, 1992, J Mol Biol 224:613-628). Although the loop comprising residues 70-75 is located on the surface of the protein and not near the active site, it appears to be affected indirectly by trisaccharide binding such that the loop shifts toward the active site and becomes relatively immobilized. The source of this loop movement appears to be the anchoring of Trp62, located in the active site cleft, as it forms a hydrogen bond with O6 of the N-acetylglucosamine at site C. Good electron density for the trisaccharide in the PEWL complex structure shows that Asp 101 is involved in hydrogen bonding interactions with the terminal sugar residue.

Crystallization and preliminary X-ray analysis of aldehyde dehydrogenase from Vibrio harveyi.

Aldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+. The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three-dimensional structure that may be correlated to cofactor specificity. Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique. In order to improve crystal size and quality, iterative seeding techniques were employed. The crystals belong to space group P2(1), with unit cell dimensions a = 79.4 A, b = 131.1 A, c = 92.2 A, and beta = 92.4 degrees. Freezing the crystal to 100 K has enabled a complete set of data to be collected using a rotating anode source (lambda = 1.5418 A). The crystals diffract to a minimum d-spacing of 2.6 A resolution. Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit. Self-rotation functions show the presence of 3 noncrystallographic twofold symmetry axes.

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications.

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.

Restructuring catalysis in the mandelate pathway.

Mandelate racemase (MR) is the first enzyme in the bacterial pathway that converts mandelic acid to benzoic acid. The mandelate pathway can utilize either enantiomer of mandelate because this enzyme interconverts them. We have solved the structure of MR at 2.5 A resolution. The enzyme is almost identical in conformation to another bacterial enzyme, muconate lactonizing enzyme (MLE). Both enzymes are TIM-barrel proteins. This result has profound implications for the evolution of enzymic function and the origin of metabolic pathways. It also implies that it should be possible to transform one enzyme into the other by site-directed mutagenesis.

Copper complexation by 3-hydroxypyridin-4-one iron chelators: structural and iron competition studies.

Clinical trials of 1,2-dimethyl-3-hydroxypyridine-4-one (1) as an orally available iron chelator are presently underway in several centers. Discrepant reports of toxicity in human and animal studies have stimulated debate on the role of iron status and the availability of iron for chelation relative to other essential elements like copper in determining the clinical effects of 1. Therefore, we investigated the ability of 1, its 1,2-diethyl analog 2, and their iron chelates to complex copper. Both compounds formed tetracoordinate 2:1 Cu(II) complexes which X-ray structure analysis showed to be planar and coordinated through the oxygen atoms of the hydroxy ketone functionality. Potentiometric analysis revealed that these complexes dominated at physiological pH, although between pH 6 and 7 approximately equal amounts of the mono and bis complexes of Cu with 1 were present at equilibrium. Comparing the stepwise formation constants deduced from the stability constants of these complexes (log beta 2 = 21.7 +/- 0.8 (1) and 20.2 +/- 2.0 (2)) with those of their Fe(III) complexes (Motekaitis,R.J.;Martell,A.E.Inorg.Chim.Acta 1991, 183,71-80) leads to a prediction of insignificant copper complexation when equimolar iron is present and dissociation products are thermodynamically unimportant. However, displacement of Fe3+ occurred from both complexes with stoichiometric amounts of Cu2+, implicating the participation of metal hydrolysis products in the equilibria. We conclude that Cu(II) complexes of the 3-hydroxypyridin-4-one chelators are stable under physiological conditions and that copper can effect displacement of iron by these agents under circumstances where hydrolysis of the metals is important.

Structure and function of S-adenosylhomocysteine hydrolase.

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1'R, 2'S, 3'R)-9-(2',3'-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor-binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17 degrees rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.

Computer simulation of aqueous biomolecular systems.

Computer simulation techniques are increasingly being used to predict structural and thermodynamic properties of large heterogeneous macromolecule and solvent assemblies. We discuss, with examples from our own studies, some problems we and others have experienced in using these techniques, which were originally devised for simple liquids. In particular, we consider the problems which arise from the large size and heterogeneity of macromolecule water systems, comparisons with experimental data and equilibrium and sampling procedures.

Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria.

The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an inner-membrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane ß-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-ß-D-N-acetylglucosamine (PNAG) secretion systems.

PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin.

The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.

Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

Structure of hexagonal turkey egg-white lysozyme at 1.65A resolution.

The structure of hexagonal turkey egg-white lysozyme (TEWL) has been determined and refined at 1.65 A resolution. The crystals were grown from a 150 mM potassium thiocyanate solution at pH 4.5 and belong to space group P6(1)22 with unit-cell dimensions a = b = 70.96, c = 83.01 A alpha = beta = 90, gamma = 120 degrees. The crystals were isomorphous with those of hexagonal pH 8.0 TEWL. The coordinates of PDB entry code 3LZ2 were therefore used as the initial model and subjected to rigid-body refinement, simulated annealing and least-squares refinement to a final residual of 0.20. The root-mean-square deviations from the ideal bond distances and angles were 0.016 A and 2.2 degrees, respectively. During the refinement, 86 water molecules and one thiocyanate ion were located in the structure. The thiocyanate ion lies close to the interface between two symmetry-related molecules. The S atom of the ion forms two direct intermolecular contacts with Argl4 and interacts indirectly via a network of water molecules to Arg5 of a symmetry-related molecule. The structure provides direct evidence for the mode of thiocyanate binding to arginine residues and suggests a possible mechanism for the efficiency of thiocyanate in crystallizing basic proteins.

Sources of funding for schools.

Public school finance mechanisms differ from state to state, and they are often extremely complex. Most commonly, the federal government contributes about 7% of the total school budget, and the remainder is split fairly evenly between local contributions (primarily raised through local property taxes) and state contributions (primarily raised through state income taxes and sales taxes). The average amount of money provided per pupil varies greatly from one state to another. The method of distributing the state contribution to school districts is equally complex, often involving some combination of basic funding (which guarantees a minimum level of general purpose support per student), power equalization (which guarantees that a certain level of local taxation will yield a given level of per-pupil funding), local option (higher levels of taxation approved in some school districts, not equalized by the state), and categorical funding (supplemental state and federal funds, earmarked for specific needs such as special education or compensatory services to schools with a concentration of poverty, or to meet state-dictated priorities, such as reducing class size or purchasing state-approved textbooks). This complexity often leads to significant variation from district to district in the percentage of funding received from federal, state, and local sources and wide disparities in the level of support for the educational program. Typically, wealthier districts provide more of their funding from local taxes, while lower-income districts are more heavily dependent on state and federal sources.

Intragenic complementation and the structure and function of argininosuccinate lyase.

Argininosuccinate lyase (ASL) catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate, a reaction important for the detoxification of ammonia via the urea cycle and for arginine biosynthesis. ASL belongs to a superfamily of structurally related enzymes, all of which function as tetramers and catalyze similar reactions in which fumarate is one of the products. Genetic defects in the ASL gene result in the autosomal recessive disorder argininosuccinic aciduria. This disorder has considerable clinical and genetic heterogeneity and also exhibits extensive intragenic complementation. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits greater enzymatic activity than is found in either of the homomeric mutant proteins. This review describes the structure and function of ASL and its homologue delta crystallin, the genetic defects associated with argininosuccinic aciduria and current theories regarding complementation in this protein.