Human Argininosuccinate Lyase: a Structural Basis for Intragenic Complementation

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9063–9068, August 1997 Biochemistry

Human argininosuccinate lyase: A structural basis for intragenic complementation

MARY A. TURNER*, ALAN SIMPSON†‡,RODERICK R. MCINNES§¶ʈ, AND P. LYNNE HOWELL*,**,††

*Division of Biochemistry Research, §Departments of Genetics and Pediatrics, and ¶Program in Developmental Biology, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada; †Laboratory of Molecular Biology and Imperial Cancer Research Fund Unit, Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom; and Departments of **Biochemistry and ʈMolecular and Medical Genetics, Faculty of Medicine, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada

Communicated by Gregory A. Petsko, Brandeis University, Waltham, MA, June 17, 1997 (received for review December 3, 1996)

ABSTRACT Intragenic complementation has been ob- Mutations in human ASL result in the clinical condition served at the argininosuccinate lyase (ASL) locus. Intragenic argininosuccinic aciduria, the second most common urea cycle complementation is a phenomenon that occurs when a mul- disorder (12). The disease displays considerable variation in its timeric protein is formed from subunits produced by different clinical pathology with three distinct phenotypes: neonatal, mutant alleles of a gene. The resulting hybrid protein exhibits subacute, and late onset (12). In an effort to understand the enzymatic activity that is greater than that found in the genetic defects that underlie argininosuccinic aciduria, oligomeric proteins produced by each mutant allele alone. The McInnes, O’Brien, and their colleagues have identified, to mutations involved in the most successful complementation date, 11 unique ASL mutations (13–15). McInnes et al. (16) event observed in ASL deficiency were found to be an aspar- also have demonstrated extensive intragenic complementation tate to glycine mutation at codon 87 of one allele (D87G) between ASL-deficient cell strains. Intragenic complementa- coupled with a glutamine to arginine mutation at codon 286 tion is a phenomenon that occurs when a multimeric protein of the other (Q286R). To understand the structural basis of is formed from subunits produced by two differently mutated alleles of a gene. Thus a partially functional hybrid protein is the Q286R:D87G intragenic complementation event at the produced from two distinct types of mutant subunits, neither ASL locus, we have determined the x-ray crystal structure of of which can, on their own, give rise to appreciable enzymatic recombinant human ASL at 4.0 Å resolution. The structure activity. has been refined to an R factor of 18.8%. Two monomers The genetic defects in the cell strains participating in the related by a noncrystallographic 2-fold axis comprise the most successful complementation event have now been iden- asymmetric unit, and a crystallographic 2-fold axis of space tified (13). The strains were found to have either a glutamine group P3121 completes the tetramer. Each of the four active to arginine mutation at codon 286 (Q286R) or an aspartate to sites is composed of residues from three monomers. Struc- glycine mutation at codon 87 (D87G). Complementation tural mapping of the Q286R and D87G mutations indicate between these alleles was demonstrated by constructing plas- that both are near the active site and each is contributed by mids expressing normal ASL, the Q286R mutant, and the a different monomer. Thus when mutant monomers combine D87G mutant and transfecting them into COS cells either randomly such that one active site contains both mutations, it individually (i.e., normal, Q286R, D87G) or together (i.e., is required by molecular symmetry that another active site both the Q286R and D87G plasmids) and measuring the rate exists with no mutations. These ‘‘native’’ active sites give rise of conversion of 14C-labeled fumarate into 14C-labeled argini- to the observed partial recovery of enzymatic activity. nosuccinate. The individual mutant D87G ASL and Q286R ASL tetramers showed little (Ϸ5%) or no (Ͻ0.01%) activity, Argininosuccinate lyase (ASL; EC 4.3.2.1) participates in the respectively, whereas the COS cells transfected with both the urea cycle, the major pathway for the detoxification of am- D87G and Q286R ASL were found to exhibit approximately monia, where it catalyzes the reversible breakdown of argini- 30% wild-type ASL activity (13). nosuccinic acid into arginine and fumarate. ASL also belongs In 1964, Crick and Orgel (17) suggested that complementation in a dimeric protein between two monomers Ab and aB to a superfamily of metabolic enzymes, all of which function as with different inactive regions (denoted by lowercase a and b) tetramers and catalyze homologous reactions with fumarate as aggregate to form an inactive site ab and an active site AB, a product. Other members of the superfamily include ␦ which results in a partial restoration of activity of Ϸ50%. crystallin (1–4), fumarase (5), aspartase (5), adenylosuccinase However, they dismissed this scenario from their general (6), and 3-carboxy-cis,cis-muconate lactonizing enzyme (7). theory of complementation, assuming that because a residual Although, across the superfamily very little sequence similarity amount of activity remained, such a protein would not be is observed, three regions of highly conserved amino acid detected as bearing mutations. In an effort to explain the sequence (5) (Fig. 1) have been identified and implicated in the observed nonlinearity of complementation maps, it was in- common catalytic mechanism. Within the superfamily, ASL is stead suggested that the ‘‘misfolding’’ of one mutant subunit most closely related to the avian eye lens protein, ␦ crystallin. was somehow compensated for by an unaltered portion of an Comparison of the protein sequences for ASL with those of adjacent mutant subunit. The three-dimensional structure of various ␦ crystallins indicate 64–72% amino acid sequence human ASL described herein has enabled us to study the identity (2–4). Certain ␦ crystallins, the ␦ II isoform, (8–11) concept of intragenic complementation found in certain ASL- have been shown to exhibit lyase activity approximately equiv- alent to that found in purified human ASL. Abbreviations: ASL, argininosuccinate lyase; TDIC, turkey ␦ I crystallin. The publication costs of this article were defrayed in part by page charge Data deposition: The coordinates and structure factors have been deposited in the Protein Data Bank, Brookhaven National Laboratory, payment. This article must therefore be hereby marked ‘‘advertisement’’ in Upton, NY, 11973 (accession nos. 1AOS and R1AOSSF, respectively). accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡Present address: Purdue University, West Lafayette, IN 47907. © 1997 by The National Academy of Sciences 0027-8424͞97͞949063-6$2.00͞0 ††To whom reprint requests should be addressed. e-mail: howell@ PNAS is available online at http:͞͞www.pnas.org. aragorn.psf.sickkids.on.ca.

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Å3⅐DaϪ1) giving rise to a solvent content of Ϸ43%. A summary of the data collection statistics is presented in Table 1. Structure Solution and Refinement. The structure of human ASL was solved by molecular replacement using the program XPLOR (29) with the coordinates of enzymatically inactive turkey ␦ I crystallin (TDIC) as the search model (30). Because the sequence of TDIC is unknown, Simpson et al. (30) have used the sequence of chicken ␦ I crystallin in their crystallographic model. ASL and chicken ␦ I crystallin share 64% amino acid sequence identity, whereas turkey and chicken are expected to share Ͼ90% sequence identity. Data between 8 and4Åresolution were used in all rotation and translational searches. Initial rotation searches and Patterson correlation refinement were carried out using coordinates of TDIC subunit A, which had lower average atomic temperature factors than the three other monomers in that structure. The search yielded two solutions with maximum rotation function values with unpruned TDIC side-chain coordinates. Subsequent translation function searches were inconclusive. In addition to TDIC (30), the structure of E. coli fumarase (31) also has been determined. The three-dimensional fold of these proteins is identical, and both associate to form a tetramer in a similar manner with almost identical 222 symmetry. This tetramer association almost certainly represents a common feature of the superfamily, and therefore it is rea- sonable to expect that the arrangement of ASL monomers would be similar. As Vm calculations indicated the presence of two monomers in the asymmetric unit of the ASL cell, a series of searches was carried out using various TDIC dimer arrange- ments. Three unique monomer combinations are possible, denoted as AB, AC, or AD. Each was used as a dimeric probe FIG. 1. The three regions of high sequence conservation among in a series of rotation searches. All rotation searches gave the members of the ASL superfamily. ASL, argininosuccinate lyase; D2C, same pair of solutions as that found in the previous searches ␦ II crystallin; fumarase, E. coli fumarase C; aspartase, E. coli aspartase; CMLE, P. putida 3-carboxy-cis,cis-muconate lactonizing using TDIC subunit A alone and after PC refinement, no enzyme; ADS, B. subtilis adenylosuccinase. dimer combination gave a significantly better result than the others. All translation searches were carried out in space deficient strains and provides experimental proof of Crick and groups P3121 and P3221. The solution was apparent as the Orgel’s initial hypothesis of intragenic complementation (17). highest peak in all translation searches at 22 SD above the Evidence for complementation according to the scheme of mean in space group P3121 for the AC dimer. This allowed the statistical regeneration of wild-type active sites also has been correct enantiomorphic space group (P3121) to be determined. The final rotation translation solution had an R factor of 39% observed in vitro for the homodimeric proteins thymidylate ͞ (R , ref. 32; 41%). synthase (18), ribulose-bisphosphate carboxylase͞oxygenase free Rigid body refinement was carried out using XPLOR with the (19), glutathione reductase (20), and mercuric reductase (21) individual monomers refined as distinct bodies to give an R and the homotrimeric enzyme aspartate transcarbamoylase factor of 34.9% and Rfree of 36.3% for data [F Ͼ 2␴(F)] between 8 (22). As intragenic complementation has been implicated in and4Åresolution. The initial model thus produced was other diseases involving mutations in homomultimeric pro- examined with the graphics package O (33), and residues were teins such as propionic acidemia (23, 24) and methylmalonic mutated computationally to the correct ASL sequence. Rig- aciduria (25), a logical extension to heteromultimeric proteins orous testing of potential XPLOR simulated annealing refine- also may apply, making intragenic complementation a much ment protocols was carried out by examination of the behavior overlooked source of phenotypic and biochemical variation in of Rfree with the aim of making the best use of the relatively genetic disease. low-resolution data. Only those protocols where both the Rfree and Rfactor decreased were considered valid. As a result of these EXPERIMENTAL METHODS tests, the decision was made to truncate the data to 4.2 Å to Crystallization and Data Collection. Recombinant human Table 1. Crystal and diffraction data ASL was expressed in Escherichia coli, purified, and crystal- Data collection lized as described previously (26). All crystals were obtained Space group P3 21 by the hanging drop vapor diffusion method. Crystals have 1 Cell dimensions a ϭ b ϭ 104.59 Å, c ϭ 183.32 Å, been grown from a 10–15 mg͞ml protein solution in 50 mM ␥ ϭ 120° phosphate buffer, 5 mM DDT at pH 7.1 with 1.1 M phosphate Resolution 4.0 Å as the precipitating agent. Intensity data were measured at Total number of reflections 23,049 room temperature using a Siemens multiwire detector Unique reflections 8,659 mounted on a Rigaku RU-200 rotating anode generator (37 Completeness* 92.3% kV, 70 mA). Intensity data were processed and scaled using the Rsym(I)† 11.6% program XDS (27, 28). On preliminary inspection, the space *Completeness quoted for all data, 69.4% of the data was Ͼ1␴(I). group was determined to be either P3 21 or its enantiomorph, 1 †Rsym(I) ϭ • (I Ϫ͗I͘)͞•Iwhere I are the intensity measurements for P3221. On the basis of density calculations two monomers were symmetry-related reflections, and ͗I͘ is the mean intensity for the estimated to be present in the asymmetric unit (Vmϭ 2.9 reflection. 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Table 2. Refinement statistics His-160, one of the putative catalytic resides. The map shows Refinement the quality of the electron density in the loop regions. The electron density for the rest of the structure was, in general, of Reflections, F Ͼ 2␴F 6,474 equal or better quality, with the exception of the electron Resolution 8–4.2 Å density for residues 71–79 and the N-terminal 19 residues. Rfactor* 18.79% These residues, therefore, have not been included in the final † Rfree 29.82% model. In addition, Glu-22 has been truncated to C␤ as have Reflections, all data 6,919 Arg-95 and Arg-246. The side chain of Arg-111 has been R factor, all data 21.16% pruned to C␣. An assessment of structure quality was made Overall B factor, Å2 20.5 using the program PROCHECK (38) and showed that no residues rms bonds, Å 0.006 rms angle, ° 1.58 resided in disallowed regions of a Ramachandran plot and that the geometry was generally excellent. Refinement results are *Rfactor ϭ • (͉Fo͉ Ϫ ͉Fc͉)͞• ͉Fo͉. listed in Table 2. †Rfree ϭ • (͉Fos͉ Ϫ ͉Fcs͉)͞• ͉Fos͉ where s refers to a subset of data not used in refinement comprising 8% of the data and selected from thin-resolution shells dispersed at intervals through the data set. RESULTS AND DISCUSSION diminish the possibility of overfitting the structure due to the Overall Topology. As expected from the close sequence incompleteness of the data in the highest resolution shell. It similarities between ASL and the delta crystallins (64–72% also proved valid to choose an appropriate weighting scheme identity) and the ASL activity demonstrated by some delta comparable to that implemented in the program PROLSQ (34) crystallins, the overall three-dimensional structures are very and based on the magnitude of the difference between ob- similar with rms deviations in dimer C␣ positions of 0.96 Å as served and calculated structure factors for ranges of sinQ͞␭ compared with TDIC (30) and 0.99 Å vs. duck ␦ II crystallin (35). This strategy resulted in lower weights for the generally (M.A.T., K. Dole, and P.L.H., unpublished work). Duck ␦ II stronger (yet more poorly modeled) lowest-resolution reflec- crystallin is a 2.0-Å resolution structure of an enzymatically tions, which otherwise would dominate a refinement run. active delta crystallin (M.A.T., K. Dole, and P.L.H., unpub- Strong noncrystallographic restraints (ncs-weight ϭ 1,500 lished work). Although ASL is a low-resolution structure, the Ϫ1 Ϫ2 kcal⅐mol ⅐Å ) were maintained during all stages of refine- simulated annealing omit trials and the general good quality of ment. This resulted in a final rms difference between noncrys- the electron density maps (see Fig. 2) allowed some significant tallographic symmetry-related monomers of 0.01 Å for all differences in the backbone atoms of the TDIC and ASL to be atoms. Several rounds of refinement were done with a simu- modeled. Each monomer is approximately 100 Å long and can lated heat stage to 4,000 K followed by cooling to 300 K and be divided into three predominantly helical domains, compris- several rounds of minimization. These rounds were alternated ing residues 1–112, 112–362, and 371–436. Fig. 3 is a schematic with sessions of rebuilding in O (33). With the individual B three-dimensional diagram of the overall topology of the factors fixed at 15 Å2, the overall B factor was refined and is monomer. ASL is catalytically active as a tetramer. Fig. 4a is listed in Table 2. Both averaged (36, 37) and unaveraged maps a schematic diagram showing the 222 symmetric arrangement with coefficients 2Fo Ϫ Fc and Fo Ϫ Fc were used during rebuilding. Extensive use of simulated annealing omit trials to of the four monomers in the tetramer. It is drawn perpendic- remove model bias and frequent comparison with the refined ular to the long axis of the helix bundle in domain 2 and viewed coordinates of two recently solved high-resolution (2.5 and 2.0 down another of the 2-fold axes. The formation of the tetramer Å) structures of two enzymatically active duck ␦ II crystallin can be described as the arrangement of two dimeric pairs mutants (M. Abu-Abed, M.A.T., C. Slingsby, and P.L.H., where each dimer is formed through mainly hydrophobic unpublished work; M.A.T., K. Dole, and P.L.H., unpublished interactions between helices h8, h11, and h12. The numbering work) aided in fitting difficult sections and confirming cor- of the helices is consistent with that described previously for rectly built regions. Refinement was completed by three TDIC (30). Less extensive interactions between dimers result rounds of positional refinement in XPLOR. Fig. 2 is a simulated in the formation of the tetramer with helix h12 of each annealing 2Fo Ϫ Fc omit map of residues 158–161, a loop monomer forming the primary, innermost interface in the between the two regions of ␤-sheet and shows the location of 20-helix bundle of the tetramer.

FIG. 2. A simulated annealing 2Fo Ϫ Fo omit map for residues 158–161. All atoms displayed in the map were removed from the structure before a round of simulated annealing refinement. The map is contoured at 1 ␴. Downloaded by guest on September 24, 2021 9066 Biochemistry: Turner et al. Proc. Natl. Acad. Sci. USA 94 (1997)

group. However, there are few other residues in the vicinity that seem capable of proton abstraction. Presumably histidine acts as a proton abstractor to form the carbanion, and cleavage is achieved upon subsequent protonation by an acid. In the ASL structure His-160 ND1 is in a position to form potential hydrogen bonds with Glu-294 OE1, Asn-289 OD1, and Pro- 290 O. The proximity of Glu-294 and its strong hydrogen bond through OE1 with the His-160 ND1 also is observed in the TDIC and fumarase structures and led Weaver et al. (31) to suggest a type of charge relay, which increases the nucleophi- licity of His-160, making it a good candidate for the role of proton abstractor. The strong hydrogen bond to Glu-294 renders His-160 less likely to carry a positive charge and therefore less likely to offer stabilization of the negatively charged intermediate. The basic residue Lys-287 is the only positively charged amino acid in this region that is strictly conserved among all superfamily members and may be responsible for neutralizing one of the three negative charges residing on the carbanion

FIG. 3. Schematic diagram showing the three-dimensional topology of an ASL monomer drawn with the program MOLSCRIPT (40). The amino acid side chains of residues D87 and Q287 have been drawn in a ball-and-stick representation. The three regions of highly conserved sequence are shaded in black.

Location of Active Site. The three regions of highly conserved sequence homology are believed, because of their intolerance to mutation, to participate in the formation of a general active site among superfamily members (30, 31). These regions are shown in Fig. 1 and are mapped onto the structure of the ASL monomer in Fig. 3. The spatial relationship between conserved regions becomes more obvious in the tetrameric arrangement of monomers in the active protein (Fig. 4a) where three different monomers contribute a section of conserved sequence to form a bowl-shaped indentation at each of the four ‘‘corners’’ of the ASL tetramer (Fig. 4 a and b). The ASL residues that contribute to the active are similar to those found in the TDIC and the E. coli fumarase structure. Further evidence that these regions comprise the active site was provided by the crystal structure of E. coli fumarase C complexed with the inhibitors tungstate and 1,2,4,5- benzenetetracarboxylic acid (31) as the inhibitor binding occurs in this putative active site pocket. Tungstate and ben- zenetetracarboxylate are thought to mimic fumarate in the enzyme complex and, by extension, may be analogous to the fumarate͞succinate moiety in an argininosuccinate complex with ASL. Earlier biochemical studies of ASL (9) implicated a histidine and a carboxylic acid as residues directly involved in catalysis. Indeed, His-160 is found at the putative ASL active site and corresponds to His-188 in fumarase C, which forms a hydrogen bond to the inhibitors through the oxygen atoms of the oxyanion. The role of His-160 in the catalytic mechanism has not yet been established. The catalytic mechanism requires a proton abstractor, a proton donor, and stabilizers of the carbanion intermediate either through interactions with the carbanion itself or through neutralization of the negatively charged carboxylates of the substrate. The side chain of FIG. 4. Schematic diagrams showing the arrangement of the His-160 could perform any of these functions depending on its conserved regions (shaded in black) in (a) the tetramer and (b) the protonation state. The structure of the fumarase C͞tungstate active site. The location of key residues are shown in a ball-and-stick inhibitor complex suggests that this histidine is involved in representation. The residues are labeled according to the monomer on bonding to and neutralizing a negatively charged carboxylate which they reside (A–D) and the residue number and type. Downloaded by guest on September 24, 2021 Biochemistry: Turner et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9067

dicarboxylate. Indeed, mutation of the corresponding residue event observed at the ASL locus yield tetrameric proteins with (Lys-326) to arginine in aspartase eliminates catalytic activity very low or no enzymatic activity, respectively. Gln-286, al- by elevating Km (39). The neutral residue Asn-289 is also though located in the loop comprising the third conserved strictly conserved and may be available for hydrogen bonding region between helices h11 and h12 (see Figs. 3 and 4b)isone to substrate. The role of Met-284 also has been speculated on of the least conserved residues across the superfamily (Fig. 1). (5) in light of absolute conservation of this residue in se- The adjacent Lys-287 has been implicated in the reaction quences of superfamily members; however, there is no exper- mechanism in light of its strict conservation throughout the imental data to suggest its possible role. The side chain of this superfamily and likely is involved in stabilizing the carbanionic residue is oriented into the active site at a distance of 11 Å from intermediate by neutralizing a negative charge either on the the His-160 side chain in the present structure. Other positively carbanion or on one the carboxylate groups of the nascent charged residues in the vicinity of the putative carboxylate fumarate. The effect of the glutamine 3 arginine mutation is binding site are Arg-113, Lys-323, and His-388, which are difficult to explain in the absence of the structure of a conserved among ASL and ␦ crystallin sequences but not complexed substrate or inhibitor molecule. The small size across the superfamily. Simpson et al. (30) speculate that difference between glutamine and arginine is unlikely to affect Glu-294, because of its proximity to His-160, is a good candi- enzymatic activity, because the glutamine side chain extends date for proton donation, whereas Weaver et al. (31) suggest upward from the loop toward the solvent and away from the for fumarase that the proton donor may be Ser-139 or Ser-140 active site. It seems more likely that the additional positive (corresponding to Ser-112 or Arg-113 of ASL). It is possible charge perturbs the mechanism in some way. Replacement of that the candidate proton donor is found in this part of the the glutamine with a positively charged arginine may enable structure; however, only two residues, Asp-115 and Thr-119, the formation of a salt bridge between Asp-367 or possibly are strictly conserved across the superfamily. Whereas Thr-119 Glu-399 of a different monomer (see Fig. 4b) resulting in a is buried and somewhat distant from the active site, Asp-115 distortion and͞or rigidification of the conserved loop, the could form a number of potential hydrogen bonds with neigh- flexibility of which may be required for activity. boring main chain atoms of Ser-112 and Glu-116 and would The D87G point mutation results in a reduction of the specific appear to be important in stabilizing the turn containing enzymatic activity to approximately 5% that of wild type. Residue residues Ser-112 and Arg-113. Asp-87 is located at the beginning of a helix in domain one (see Location of Q286 and D87. The D87G and Q286R muta- Figs. 3 and 4b) and is in close proximity to His-89, a residue we tions associated with the most sucessful complementation postulate as being important for the binding of the substrate and

FIG. 5. A pictorial representation of the active sites of the statistically available combinations of mutants in D87G:Q286R intragenic complementation of the ASL tetramer. For clarity the diagram has been drawn to show the interaction of only the D87 (ovals) and Q286 (circles) and the shading of the symbols represents the presence of the point mutations D87G and Q286R, respectively. Each large circle represents one of the four active sites found in the protein. In the schematic diagram of the native protein, residues 286 and 87 also are labeled according to which monomer they are found on. The monomers are designated A-D. Due to the molecular symmetry of the tetramer, in the case of the 2D87G:2Q286R tetramer there are three distinctly different ways of combining the monomers that will give rise to either two or zero native active sites being formed. For each combination of monomers, one also must consider which monomer contains the mutation, in the case of 3D87G:1Q286R tetramer, the Q286R mutation could be present on monomer A, B, C, or D, and this gives rise to four different possible solutions. The set of all possible combinations has a degeneracy corresponding to a binomial distribution of 1:4:6:4:1. Downloaded by guest on September 24, 2021 9068 Biochemistry: Turner et al. Proc. Natl. Acad. Sci. USA 94 (1997)

in defining the substrate specificity of the enzyme (M. Abu-Abed, 6. Stone, R. L., Zalkin, H. & Dixon, J. E. (1993) J. Biol. Chem. 268, M.A.T., C. Slingsby, and P.L.H., unpublished work). Side-chain 19710–19716. density is weak for residues in this region, implying some disorder 7. Williams, S. E., Woolridge, E. M., Ransom, S. C., Landro, J. A., due to flexibility; however, potential hydrogen bonds could be Babbitt, P. C. & Kozarick, J. W. (1992) Biochemistry 31, 9768– formed between Asp-87 and main chain of Thr-90 and between 9776. 8. Barbosa, P., Wistow, G. J., Cialkowski, M. J. P. & O’Brien, W. E. Asp-87 and Glu-86 (Å). This residue therefore could play a role (1991) J. Biol. Chem. 266, 22319–22322. in stabilizing the helix by hydrogen bonding to the nitrogen of the 9. Lee, H.-J., Chiou, S.-H. & Chang, G.-G. (1992) Biochem. J. 283, main chain of Thr-90 (i.e., N-capping this helix). This hydrogen 597–603. bonding capability would be lost upon mutation to glycine as 10. Chiou, S.-H., Lo, C.-H., Chang, C.-Y., Itoh, T., Kaji, H. & would the negative charge, both of which may play a role in Samejma, T. (1991) Biochem. J. 273, 295–300. enzyme structure stabilization or in formation of the enzyme͞ 11. Kondoh, H., Araki, I., Yasuda, K., Matsubasa, T. & Mori, M. substrate complex. (1991) Gene 99, 267–271. Complementation in Hybrid Tetramer. Whereas the struc- 12. Brusilow, S. W. & Horwich, A. L. (1995) in The Metabolic Basis ture of ASL presented here is a low-resolution structure and of Inherited Disease, eds. Scriver, C. R., Beaudet, A. L., Sly, W. S. the side-chain conformations are less precisely defined than & Valle, D. (McGraw–Hill, New York), 6th Ed., Vol. 1, pp. they would be in a high-resolution structure, it is, however, 1187–1232. 13. Walker, D. C., Christodoulou, J., Craig, H. J., Simard, L. R., obvious that the individual mutations, D87G and Q286R, Ploder, L., Howell, P. L. & McInnes, R. R. (1997) J. Biol. Chem. which participate in most successful complementation event 272, 6777–6783. observed at the ASL locus, are located at different loci in the 14. Barbosa, P., Cialkowski, M. & O’Brien, W. E. (1991) J. Biol. active site cleft (Fig. 3b) and thus detrimentally affect different Chem. 266, 5286–5290. aspects of the catalytic mechanism. Any active site containing 15. Walker, D. C., McCloskey, D. A., Simard, L. R. & McInnes, R. R. one or the other or both mutations would be compromised. (1990) Proc. Natl. Acad. Sci. USA 87, 9625–9629. Because a monomer will contain only one of the point 16. McInnes, R., Shih, V. & Chilton, S. (1984) Proc. Natl. Acad. Sci. mutations, there are five statistically distinguishable monomer USA 81, 4480–4484. combinations that can be formed. These unique combinations 17. Crick, F. H. C. & Orgel, L. E. (1964) J. Mol. Biol. 8, 161–165. can give rise to the following tetramers in D87G:Q286R 18. Pookanjanatavip, M., Yuthavong, Y., Greene, P. J. & Santi, D. V. (1992) Biochemistry 31, 10303–10309. polypeptide ratios 4:0, 3:1, 2:2, 1:3, and 0:4. Examination of 19. Larimer, F. W., Lee, E. H., Mural, R. J., Soper, T. S. & Hartman, each of the four active sites in the protein reveals that for any F. C. (1987) J. Biol. Chem. 262, 15327–15329. one monomer (e.g., monomer A), residues 87 and 286 are 20. Scrutton, N. S., Berry, A., Deonarain, M. P. & Perham, R. N. found in two different active sites (active sites 1 and 2 in Fig. (1990) Proc. R. Soc. London Ser. B 242, 217–224. 5). The second half of the diagram illustrates the results for the 21. Distefano, M. D., Moore, M. J. & Walsh, C. T. (1990) Biochem- four active sites for each of the various possible tetramer istry 29, 2703–2713. combinations. When the set of all possible hybrid tetramer 22. Wente, S. R. & Schachman, H. K. (1987) Proc. Natl. Acad. Sci. combinations is considered there is a degeneracy correspond- USA 84, 31–35. ing to the binomial distribution 1:4:6:4:1 to give an overall 23. Gravel, R., Akerman, B. R., Lamhonwah, A.-M., Loyer, M., expected enzymatic activity of Ϸ25% of that of the wild-type Leon-del-Rio, A. & Italiano, I. (1994) Am. J. Hum. Genet. 55, 51–58. protein, assuming that zero activity is observed for each of the 24. Gravel, R. A., Lam, K. F., Scully, K. J. & Hsia, Y. (1977) Am. J. homotetrameric mutants and that the monomers combine in a Hum. Genet. 29, 378–388. randomly distributed manner. This expected level of activity is 25. Qureshi, A. A., Crane, A. M., Matiaszuk, N. V., Rezvani, I., close to the 30% of wild-type activity seen in the reconstruc- Ledley, F. D. & Rosenblatt, D. S. (1994) J. Clin. Invest. 93, tion of the complementation event in COS cells (13), the 1812–1819. difference between the two values being accounted for by the 26. Turner, M. A., Achyuthan, A. M., McInnes, R. R., Hershfield, M. 5% activity exhibited by the D87G tetramer. & Howell, P. L. (1994) J. Mol. Biol. 239, 336–338. Although the exact roles of Q286 and D87 are still in 27. Kabsch, W. (1988) J. Appl. Cryst. 21, 67–71. question, the structure of ASL has provided experimental 28. Kabsch, W. (1988) J. Appl. Cryst. 21, 916–924. proof that Crick and Orgel’s (17) initial intragenic comple- 29. Bru¨nger, A. T. (1993) XPLOR Version 3.1 (Yale Univ. Press, New Haven). mentation hypothesis regarding the statistical regeneration of 30. Simpson, A., Bateman, O., Driessen, H., Lindley, P., Moss, D., wild-type active sites was correct. Furthermore, it appears Mylvaganan, S., Narebor, E. & Slingsby, C. (1994) Nat. Struct. likely that the phenomenon of intragenic complementation is Biol. 1, 724–733. as yet an unappreciated source of phenotypic variation in 31. Weaver, T. M., Levitt, D. G., Donnelly, M. I., Wilkens Stevens, genetic diseases involving multimeric proteins. P. P. & Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 654–662. 32. Brunger, A. T. (1992) Nature (London) 355, 472–474. We thank M. S. Hershfield for providing the purified protein for the 33. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991) initial crystallization trials and the ASL plasmid, C. Slingsby for Acta Crystallogr. A 47, 110–119. providing the turkey ␦ I crystallin coordinates, and G. D. Smith for his 34. Hendrickson, W. A. & Konnert, J. H. (1980) in Incorporation of help with the weighting schemes used in the crystallographic refine- Stereochemical Information into Crystallographic Refinement, eds. ment and comments on the manuscript. This work is supported by a Diamond, R., Ramaseshan, S. & Venkatesan, K. (International grant from the Natural Science and Engineering Research Council of Union of Crystallography, Bangalore, India), Vol. 13, pp. 1–23. Canada to P.L.H. R.R.M. is an International Research Scholar of the 35. Smith, G. D. (1997) Acta Crystallogr. D52, 41–48. Howard Hughes Medical Institute. 36. Kleywegt, G. J. & Jones, T. A. (1994) in Halloween...Masks and Bones, eds. 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