Human Molecular Genetics, 2001, Vol. 10, No. 6 635-643
© 2001 Oxford University Press
Mutations in the regulatory domain of cystathionine ß–synthase can functionally suppress patient-derived mutations in cis
1Division of Population Science and 2Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
Received 27 December 2000; Revised and Accepted 29 January 2001.
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ABSTRACT |
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Human cystathionine ß–synthase (CBS) is an S-adenosylmethionine-regulated enzyme that plays a key role in the metabolism of homocysteine. Mutations in CBS are known to cause homocystinuria, an inborn error in metabolism. We previously developed a yeast functional assay for CBS and used it to characterize mutations found in homocystinuric patients. We discovered that many patient-derived mutations are functionally suppressed by deletion of the C-terminal 142 amino acids, which contain a 53 amino acid motif known as the CBS domain. This domain is found in a wide variety of proteins of diverse biological function. Here we have used a genetic screen to identify missense mutations in the C-terminal region of CBS that can suppress the most common patient mutation, I278T. Seven suppressor mutations were identified, four of which map to the CBS domain. When combined in cis with another pathogenic mutation, V168M, six of seven of the suppressor mutations rescued the yeast phenotype. Enzyme activity analyses indicate that the suppressors restore activity from <2% to 17–64% of the wild-type levels. Analysis of the suppressor mutations in the absence of the pathogenic mutation shows that six of the seven suppressor alleles have lost enzymatic responsiveness to S-adenosylmethionine. Using homology modeling, we show that the suppressor mutations appear to map on one face of the CBS domain. Our results indicate that subtle changes to the C-terminus of CBS can restore activity to mutant proteins and provide a rationale for screening for compounds that can activate mutant CBS alleles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the cystathionine ß–synthase (CBS) gene are the most common cause of homocystinuria, a genetic disorder characterized by extremely elevated levels of total homocysteine (tHcy) in plasma (1). Homocystinuric individuals have a variety of clinical phenotypes including mental retardation, ectopia lentis, skeletal abnormalities and thrombotic vascular disease. About half of CBS-deficient patients respond to pharmacological doses of pyridoxine (vitamin B6) with significant lowering of tHcy and a reduction in the severity of disease phenotypes.
The human CBS enzyme has been extensively characterized. It condenses homocysteine with serine to form cystathionine, which is then used to form cysteine. The enzyme is 551 amino acids in length, forms a homotetramer of 63 kDa subunits and requires pyridoxal phosphate and heme for activity (2–4). In addition, enzyme activity is stimulated 2–3-fold by the addition of S-adenosylmethionine (Adomet) (5). Previous work from our and other laboratories has established a modular structure of the CBS enzyme, where the catalytic domain is located in the N-terminal 409 amino acids and a regulatory domain is located in the C-terminal 142 amino acids (6,7). This regulatory domain is required for activation by Adomet and for Adomet binding (8). The truncated enzyme appears to be constitutively active, suggesting that Adomet activation works by inhibiting the inhibitory effects of the C-terminal domain. Interestingly, within the C-terminal domain is a highly conserved 53 amino acid motif, which has been dubbed the ‘CBS domain’ (9). This motif is present in a variety of unrelated proteins including inosine monophosphate dehydrogenase (IMPDH), 5'AMP-activated protein kinase, chloride channels and a variety of other proteins. In all proteins except CBS this motif is present in multiple copies. No function of this domain has yet been described. The three-dimensional structure of the CBS domains in IMPDH from Streptococcus pyogenes has been determined to high resolution (10).
In previous work, our laboratory described an unexpected finding regarding the relationship between the C-terminal regulatory domain of CBS and missense mutations found in homocystinuric patients. We found that the activity of 9 of 10 patient-derived alleles was restored by deleting the C-terminus, even though these mutations all mapped within the catalytic domain (6). Thus, most patient-derived mutations in CBS seem to affect CBS regulation, not catalysis.
These results suggest that it might be possible to develop drugs to treat CBS deficiency by targeting the C-terminal domain. To explore this possibility further we have developed a genetic selection in yeast to search for missense mutations in the C-terminal domain that can suppress patient-derived mutations in cis. In this report we describe and characterize seven missense mutations discovered in this screen. Our ability to isolate such mutations suggests that even subtle alterations in the C-terminal domain can activate mutant CBS, further strengthening the idea that the C-terminal domain of CBS may be a good therapeutic target for CBS deficiency. Furthermore, four of the seven suppressor mutations map to the 53 amino acid CBS domain, suggesting that this domain may play a key role in the regulation by Adomet.
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RESULTS |
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Suppression of disease-causing CBS mutations by C-terminal mutations
Previously we have shown that expression of wild-type human CBS can complement the Cys– phenotype of the yeast CBS deletion strain, WY35, whereas expression of the I278T mutant form of the enzyme cannot. In order to isolate C-terminal missense mutations that could suppress I278T in cis, we set up a screen combining random PCR mutagenesis with homologous recombination in yeast (Fig. 1). We screened 2500 colonies for C-terminal suppressors of I278T and found seven suppressors based on their ability to allow growth on Cys– media. The CBS-expressing plasmids in these strains were retrieved and sequenced and the locations of the suppressor mutations were determined (Fig. 2). All of the plasmids analyzed contained a single mutation in the C-terminal region of CBS. Four of the seven mutations mapped within the CBS domain (T424N, I429N, I437T and G453E) and three mapped outside this region (H513P, R527W and G532E). All seven of the suppressor mutations allowed yeast to form single colonies on Cys– media, although some grew slightly more slowly than yeast containing wild-type CBS (Fig. 3).
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Our previous work had shown that several different missense alleles of CBS could be suppressed in cis by deletion of the C-terminus. Therefore, we examined the ability of our missense suppressors to suppress a second CBS allele, V168M. All seven of the missense suppressors were combined in cis with V168M using double-gap repair (Materials and Methods) and tested for growth in the absence of cysteine. Six of the seven suppressors (H513P being the exception) allowed growth (Fig. 3). Thus, like C-terminal truncation, the C-terminal missense suppressors can suppress multiple alleles.
We next examined whether the C-terminal mutations affect CBS expression levels. Western blot analysis of yeast whole- cell extracts using anti-hCBS antibody demonstrated that all the suppressors in combination with I278T were expressed at levels comparable to wild-type CBS (Fig. 4). The suppressors in combination with V168M also expressed full-length CBS, but there appeared to be somewhat reduced levels of all the proteins and some small amount of proteolysis. Similar proteolysis was observed in the purification of V168M from bacteria (Fig. 4) (6). However, these data clearly show that the suppression phenotype is not due to overexpression of the double mutant proteins.
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Enzyme activity
We next tested CBS enzyme activity in total yeast extracts in the presence and absence of Adomet. Extract from yeast expressing wild-type human CBS contained 186 mU of activity in the absence of Adomet and 487 mU in the presence of Adomet, a 2.6-fold increase (Fig. 5). Extracts from yeast expressing the I278T allele of CBS had undetectable levels of activity (<2% of wild-type). The activities of extracts from strains expressing I287T in combination with the C-terminal suppressor mutations ranged from 32 to 119 mU, or 17 to 64% of wild-type activity. Interestingly, the double mutant proteins showed markedly reduced or no stimulation of activity by Adomet. Only the I278T/H513P combination showed a 2-fold or better stimulation with Adomet. These studies show that the suppressor mutations can restore significant enzymatic function to an essentially inactive enzyme.
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We also examined enzyme activity of the suppressor mutations in the absence of the upstream pathogenic change. Using plasmid gap repair we engineered all of the suppressor mutations into wild-type CBS. All of the resulting yeast strains grew well on media lacking cysteine, indicating that they must have CBS activity (data not shown). Activity in total yeast extracts was also examined. Two of the mutants, I437T and G453E, had activity comparable to wild-type CBS, although there was no stimulation by Adomet. Four other mutants, T424N, I429N, R527W and G532E, were somewhat less active than wild-type CBS and also exhibited no Adomet stimulation. One mutation, H513P, showed significant Adomet stimulation. These studies show that most missense mutations in the C-terminus block activation by Adomet.
Molecular modeling of the CBS domain in human CBS
The CBS domain-containing protein IMPDH from S.pyogenes has significant sequence similarity to human CBS as determined by PSI-BLAST (Fig. 6A). S.pyogenes IMPDH contains two CBS domains, the second of which (163–212) shares 20% identity and 45% similarity with amino acids 421–470 of CBS. In addition, secondary structure prediction, using PSIPRED (11), indicates that this region of CBS has a similar 
-helix/ß-sheet pattern to IMPDH. Specifically, both appear to have the structure E1-H1-E2-E3-H2, where E stands for ß-sheet and H for -helix.
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Given the evidence that these two regions shared similar structure, we used homology modeling to build a three-dimensional model of human CBS residues 421–470, using the IMPDH amino acids 163–212 as a template. This model incorporates amino acids containing four of the suppressor mutations isolated (Fig. 7A). According to the model, all four mutations lie on the same face of the CBS domain. This face is hydrophobic in nature and three of the four alterations, I429N, I437T and G453E, convert hydrophobic or neutral residues to polar ones. In the middle of this hydrophobic patch is a single polar residue, threonine 424, which is mutated to a more polar asparagine. These observations are consistent with the hypothesis that this region may be involved in protein–protein contacts.
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CBS is unusual in that all other CBS domain proteins contain at least two copies of the motif, whereas CBS itself only has one. Therefore we have carefully examined the CBS sequence to see if there may in fact be a second CBS domain. Using PSI-BLAST we were able to detect weak similarity with both CBS domains in S.pyogenes IMPDH. This alignment is shown in Figure 6B. Note that in this alignment the ‘strong’ CBS domain in human CBS is aligned with the first CBS domain IMPDH, and that there appears to be a weak second domain extending from 486 to 543. A secondary structure prediction with PSIPRED based on a multiple sequence alignment of CBS orthologs is also shown above the alignment in Figure 6B. While the ß-sheet strands are not well predicted, the
-helices are in reasonable agreement with the crystal secondary structure shown below the alignment. It should be noted that this alignment is less statistically significant than the alignment and model shown in Figure 6A and should be treated with caution. Using this second alignment we have created a second structural model (Fig. 7B). This model shows how the two CBS domains might interact. This two-CBS domain model allows us to map all seven suppressor mutations isolated. Six of the seven suppressor mutations appear to lie on the same face of the domain and are either in or adjacent to a strong hydrophobic surface in the modeled structure. Five of the suppressor mutations, T424N, I429N, I437T, G453E and G532E, would be predicted to make this surface more hydrophilic.
We also modeled the mutations themselves into the structure to see if the mutant amino acids could be accommodated within the predicted structure (Materials and Methods). Based on this analysis, there appears to be enough space for all the side-chain changes with the exception of H513P. The proline appears to bump into a phenylalanine (F531), although it is located in a loop constructed in the modeling process so it could potentially move out of the way. However, a H
P mutation is a significant change that might alter the conformation of this loop.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In earlier work our laboratory demonstrated that deletion of the C-terminal regulatory domain of CBS could at least partially suppress the functional effects of missense mutations located in the catalytic domain (6). In addition we showed that truncated CBS was constitutively upregulated, and no longer responded to the allosteric regulator Adomet. In this paper we demonstrate that it is possible to activate mutant CBS enzymes by point mutations in the C-terminal regulatory domain. Like the C-terminal deletion allele, these missense suppressors are able to restore enzymatic function to molecules inactivated by two different missense mutations in the catalytic domain. Also, most of the missense suppressors, with the exception of H513P, are no longer able to respond to stimulation of Adomet. However, in contrast to the deleted form of CBS, the C-terminal mutant proteins are not constitutively upregulated. Several of the suppressors have activity levels similar to unstimulated wild-type CBS. However, none has levels equivalent to the Adomet stimulated level, which is in contrast to C-terminal deleted CBS (7).
Our observations have led to a model of CBS function, illustrated in Figure 8. In this model the regulatory domain of wild-type CBS in the absence of Adomet inhibits CBS activity, perhaps by partially blocking access to and from the active site. Kinetic and biochemical studies of recombinant human CBS indicate that upon exposure of the enzyme to Adomet the enzyme undergoes a conformational change that causes an increase in the turnover rate of the enzyme (7). In the absence of the regulatory domain, access to the active site is unimpaired and the enzyme is then constitutively active. Our observation that missense mutations can be suppressed by C-terminal truncations and mutation suggests that most disease-causing missense mutations work by locking the enzyme in a closed conformation, perhaps by altering how the regulatory domain interacts with the catalytic domain. Deletion of the regulatory domain would eliminate this unproductive interaction and allow the catalytic domain to be accessible to the substrate.
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We hypothesize that the missense mutations identified here alter the conformation of the regulatory domain such that it can no longer undergo a conformational change in response to Adomet, essentially locking it in a partially open conformation. This could explain why the missense suppressors are less active than the deletion mutation: the deletion mutation is totally open, whereas the missense suppressor is only partially open. In combination with disease-causing missense mutations like I278T, the suppressor missense mutations could alter the regulatory domain structure in such a way that it cannot be locked into a closed conformation by I278T.
For simplicity this model does not include the fact that CBS is a tetramer. It is possible that the regulatory domain of one subunit actually interacts with the catalytic domain of a different subunit. Interestingly, in some dimeric enzymes, like thymidylate synthase, when substrate is bound to one subunit this causes a conformational change that inhibits substrate binding to the other subunit (12). After catalysis is complete a conformational change occurs altering the dimer interface such that the other subunit can then bind substrate. Thus catalysis at each site occurs in alternation. Such enzymes are referred to as half-site active. It has been demonstrated that CBS tetramer contains two non-equivalent sites with regards to heme and PLP binding (13), consistent with a half-site active mechanism. If this is the case, patient-derived mutations that are suppressible by C-terminal truncation or mutation may be defective in causing or transmitting the conformational change necessary for activation.
Four of the seven suppressor mutations described here map to the canonical CBS domain located in CBS. The structure of this domain can be inferred from the crystal structure of the CBS domain in IMPDH. Assuming that that the CBS domain in CBS has a similar structure, it appears that all four of the suppressor mutations that map to this domain are located on one face of the structure. This face is very hydrophobic and three of the four alterations convert hydrophobic residues to polar ones. This suggests that these mutations could disrupt the interaction of this domain with another part of the CBS protein. More speculative is the view that CBS might actually contain a second CBS domain. There is weak sequence similarity between the C-terminal regulatory domain of CBS and both CBS domains of IMPDH when aligned together. If the CBS sequences are modeled using this alignment, six out of seven of the suppressor mutations are on the same side of the structure, either in or adjacent to a large hydrophobic patch. Taken together, modeling supports the idea that the suppressor mutations may alter how the regulatory domain interacts with the catalytic domain.
Our work also demonstrates that subtle alterations in the regulatory domain can restore function to mutant proteins found in patients with homocystinuria. All of the suppressor mutations were able to suppress the I278T alteration in CBS. I278T is the most common mutation found in CBS-deficient patients throughout the world (14). Although the majority of patients with I278T are pyridoxine responsive, clearly some are non-responsive (15). It should also be noted that even in responsive patients, plasma homocysteine tends to remain elevated at levels three to four times that of healthy individuals. Our data suggest it may be possible to identify compounds that interact with the C-terminal domain of CBS such that they mimic the effect of the missense mutations described here. Such compounds may make useful drugs in the treatment of CBS deficiency. Although we have only tested the ability of the suppressors isolated here to suppress two disease-causing mutations, I278T and V168M, in previous work we have shown that truncation of CBS can restore enzyme activity to a majority of patient-derived mutant enzymes (6). Thus, it may be possible that compounds that target the C-terminal domain may be generally useful in treating CBS-deficient patients.
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MATERIALS AND METHODS |
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Strains and reagents
Yeast strain WY35 (MAT
ura3-52 his3 trp1 cys4::LEU2) and plasmids pHCBS
, pI278T and pV168M were created as described by Kruger and Cox (16). General yeast methods were performed as described by Sherman (17).
PCR-based random mutagenesis and screening
Plasmid pHCBS was used as a template (3). Primers 5'-gtggcagtgctggcagcacg-3' (forward) and 5'-gaggaaagcgaaggagaagtgggca-3' (reverse) were used to amplify a fragment coding for CBS amino acids 348–551 under conditions that enhance PCR error rates described by Fromant et al. (18).
The N-terminal fragment containing the I278T mutation was created in the following manner. Plasmid pI278T was used as a template for PCR using primers 5'-atgccttctgagaccccccaggcag-3' (forward) and 5'-atgtagttccgcactgagtc-3' (reverse). This PCR fragment encodes amino acids 1–381. The PCR was performed using Pfu polymerase to reduce the possibility of PCR error.
A two-fragment gap-repair procedure was used to create a library of double-mutant CBS molecules. The gap-repair vector pHCBS was prepared as described by Kruger and Cox (16). Prepared vector (10 ng) mixed with 
0.5 µg of each of the two PCR products described above was used to transform yeast strain WY35. Transformants were selected on SC-Trp media supplemented with glutathione. Transformants were then replica-plated to SC-Trp plates lacking glutathione. Seven colonies were identified that could grow in the absence of glutathione (the source of cysteine).
Colonies that grew in the absence of glutathione were reisolated and confirmed by streaking on SC-Trp plates. Cells were then grown in liquid media (SC-Trp) and total yeast DNA was isolated. From this DNA, the entire CBS open reading frame (ORF) was PCR-amplified and sequenced.
Construction of other mutant CBS molecules
Plasmids containing the suppressor mutations in cis with V168M were created as follows. Total yeast DNA obtained from yeast strain WY35 containing pV168M was used as template for Pfu-driven PCR with primers used to amplify N-terminal sequences as described above. The CBS C-terminal fragment was derived by Pfu-driven PCR from total yeast DNA from the suppressor mutant-containing strains. The two PCR products were then mixed with linearized pHCBS and used to transform WY35. DNA was isolated from one transformant containing each construct and the CBS ORF was PCR-amplified and sequenced to confirm that the proper mutations were present.
Plasmids containing the suppressor mutants in isolation were made identically as described above except that the N-terminal PCR fragment was derived from pHCBS (3).
Crude yeast extracts
Crude yeast extracts were made by mechanical lysis of desired yeast strain grown to an optical density of 0.5–1 at 30°C. Briefly, yeast cells were resuspended in a lysis buffer (50 mM Tris pH 6.8, 100 mM NaCl, 1 mM PMSF) after harvesting. The cell suspension was kept on ice and 0.5 mm glass beads were added. The cells were lysed using a Beadsbeater (Biospec Products). The lysate was checked under a microscope to ensure 80–90% breakage. Glass beads and cell debris were removed by centrifugation. To remove the endogenous Adomet, the extract was dialyzed against the lysis buffer for 10 h in a Micro DispoDialyzer (Spectrum). Protein concentrations were determined using the BCA Protein Assay kit from Pierce.
Immunoblot analysis
Immunoblot assay was carried out according to standard protocols (19). About 150 µg of total protein was loaded in a well of a 10% SDS–PAGE gel. The proteins were transferred to a nitrocellulose membrane and probed with purified anti-CBS antibody. The secondary antibody used was alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad). Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were used as developing regents.
In vitro CBS enzyme activity assay
CBS activity was assayed essentially as described (20). An 18 µg reaction volume containing 50 µg of yeast extract was incubated in 200 mM Tris–Cl pH 8.6, 250 µM pyridoxal phosphate, 800 µM cystathionine and 5 mM L-14C-serine (800 c.p.m./nmol). In reactions including Adomet it was added to a final concentration of 100 µM. The reaction was initiated by the addition of 2 µl of 50 mM homocysteine. After incubation 1 for 1 h at 37°C, the reaction was terminated by the addition of 5 µl of 50% trichloroacetic acid and the protein was precipitated. Five microlitres of the supernatant was then spotted on thin layer chromatography (TLC) cellulose plates (Selecto Scientific) and separated by ascending TLC in 2-propanol/formic acid/H2O (80/6/20 v/v). Radioactivity was then quantitated using a PhosphorImager (Fuji 1000). Milliunits are expressed as nmol of cystathionine produced per milligram of total yeast protein per hour.
Structural modeling
We used the methods described previously by Dunbrack (21) to build a model of the CBS domain. Briefly, PSI-BLAST (22) was used to build a sequence profile of the CBS domain family by iteratively searching the non-redundant protein sequence database available from NCBI. We used four iterations of PSI-BLAST, and only sequences with expectation values better than 0.0001 were included in the sequence profile matrix. We used the program PSIPRED (11) to predict the secondary structure of this region. PSIPRED uses a multiple sequence alignment produced by PSI-BLAST as input. Upon completion, this matrix was used to search a database of sequences of proteins in the Protein Data Bank (PDB) (23) of experimentally determined protein structures. This resulted in a list of three IMPDH proteins from three organisms that could be used as a template for modeling the CBS domain. The highest resolution structure was a 1.9 Å crystal structure of IMPDH from S.pyogenes, PDB entry 1ZFJ (10). The alignment of CBS and 1ZFJ was adjusted manually to remove two small gaps near the ends of the domain. This resulted in an alignment with no gaps, as shown in Figure 6A. The backbone of the model was constructed with the program blast2model (J.M. Sauder and R.L. Dunbrack, unpublished data; http://www.fccc.edu/research/labs/dunbrack/software). Side chains were constructed with the side-chain modeling program SCWRL (21,24), which uses a backbone-dependent rotamer library (25–27) followed by a dead-end elimination and branch-and-bound search to remove steric overlaps. Conserved side chains according to the alignment in Figure 6A were kept in their Cartesian coordinates from PDB entry 1ZFJ.
PSI-BLAST searches also produced alignments of residues 402–543 of CBS to IMPDH PDB entry 1ZFJ residues 78–210. This region covers both CBS domains of IMPDH. This led us to conclude tentatively that there may be a second CBS domain in CBS. However, a PSI-BLAST search with residues 471–551 of CBS as well as a threading search with the program THREADER (28) did not produce an alignment with IMPDH or other CBS domains from non-CBS proteins. To explore this further, we used the program PSIPRED (11) to predict the secondary structure of this region. Since the long alignment might be an artifact of the strong CBS domain signal in the region of 421–470 of CBS, we used only orthologous sequences of human CBS in the alignment used as input for PSIPRED. This secondary structure prediction is shown in Figure 6B in the alignment of residues 421–551 of CBS against residues 100–218 of IMPDH. The alignment shows that some of the secondary structure units of IMPDH are reproduced in the secondary structure prediction of CBS in both the first CBS domain and the tentative second CBS domain. We therefore provide the alignment and model of two CBS domains in Figures 6B and 7B as a hypothesis requiring experimental verification.
The model of the CBS domain pair was produced in a three- step procedure. First, the sequence of CBS was placed on the backbone of IMPDH residues 100–218 according to the alignment in Figure 6A using the SCWRL program. At this stage, the insertions and deletions in the alignment were left out of the model. Second, the recently developed loop-building routine (29) in the MODELLER program (30) was used to build insertions in the CBS model and to close up deletions. The coordinates of all amino acids except each modeled loop and three residues on either side of the gap were held fixed, while the loop was constructed according to a simulated annealing and energy minimization protocol within MODELLER. Third, all side chains in the model were rebuilt with SCWRL, except those conserved in the alignment in Figure 6A, which were held fixed.
We also built models of the mutants described in the experiments. The side chains in the final model of the CBS domain pair (Fig. 7A) were rebuilt and the mutated side chain was replaced with the mutant allele. Again, conserved side chains in the alignment were held fixed in their Cartesian coordinates from IMPDH.
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ACKNOWLEDGEMENTS |
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R.L.D. thanks the American Cancer Society for computer support. This study was aided by grants from the Bugher Foundation, USPHS grants HL57299-01 and CA06927 from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Fox Chase Cancer Center, P3016, 7701 Burholme Avenue, Philadelphia, PA 19111, USA. Tel: +1 215 728 3030; Fax: +1 215 214 1623; Email: wd_kruger@fccc.edu
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