Crystal Structure of T-protein of the Glycine Cleavage SystemCOFACTOR BINDING, INSIGHTS INTO H-PROTEIN RECOGNITION, AND MOLECULAR BASIS FORUNDERSTANDING NONKETOTIC HYPERGLYCINEMIA*

Received for publication, August 23, 2004, and in revised form, September 3, 2004Published, JBC Papers in Press, September 7, 2004, DOI 10.1074/jbc.M409672200

Hyung Ho Lee‡§, Do Jin Kim‡§, Hyung Jun Ahn§, Jun Yong Ha, and Se Won Suh¶

From the Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea

The glycine cleavage system catalyzes the oxidativedecarboxylation of glycine in bacteria and in mitochon-dria of animals and plants. Its deficiency in humancauses nonketotic hyperglycinemia, an inborn error ofglycine metabolism. T-protein, one of the four compo-nentsoftheglycinecleavagesystem,isatetrahydrofolate-dependent aminomethyltransferase. It catalyzes thetransfer of the methylene carbon unit to tetrahydrofo-late from the methylamine group covalently attached tothe lipoamide arm of H-protein. To gain insight into theT-protein function at the molecular level, we have de-termined the first crystal structure of T-protein fromThermotoga maritima by the multiwavelength anoma-lous diffraction method of x-ray crystallography andrefined four structures: the apoform; the tetrahydrofo-late complex; the folinic acid complex; and the lipoicacid complex. The overall fold of T-protein is similar tothat of the C-terminal tetrahydrofolate-binding region(residues 421–830) of Arthrobacter globiformis dimeth-ylglycine oxidase. Tetrahydrofolate (or folinic acid) isbound near the center of the tripartite T-protein. Lipoicacid is bound adjacent to the tetrahydrofolate bindingpocket, thus defining the interaction surface for H-pro-tein binding. A homology model of the human T-proteinprovides the structural framework for understandingthe molecular mechanisms underlying the developmentof nonketotic hyperglycinemia due to missense muta-tions of the human T-protein.

The glycine cleavage system (GCS)1 serves an importantbiochemical function by catalyzing the oxidative decarboxyla-tion of glycine in the mitochondria of animals and plants aswell as in bacteria (1). It is composed of four components:P-protein (EC 1.4.4.2); H-protein; T-protein (EC 2.1.2.10); andL-protein (EC 2.1.8.1.4). Inherited deficiency of the humanmitochondrial GCS causes nonketotic hyperglycinemia (NKH),

an inborn error of glycine metabolism (2, 3). It is characterizedby elevated levels of glycine in blood and cerebrospinal fluid. Inpatients with NKH, convulsive seizures, coma, and respiratorydistress develop within a few days after birth (4). A defect inany component of the GCS can abolish the overall activity ofthe GCS. Up to 15% NKH patients have defects in T-protein,and most other patients have P-protein defects, whereas H-protein and L-protein deficiencies are rare (5).

P-protein of the GCS catalyzes the pyridoxal phosphate-de-pendent decarboxylation of glycine and transfer of the residualmethylamine moiety to the lipoyl-lysine arm of the oxidizedH-protein, generating a methylamine-loaded H-protein. Whenmethylamine-bound, the lipoamide arm of H-protein is pivotedand is tightly bound into a cleft at the protein surface (6).T-protein is a tetrahydrofolate (H4folate)-dependent amino-methyltransferase. It catalyzes transfer of the methylene car-bon unit to T-protein-bound H4folate from the methylaminegroup covalently attached to the lipoamide arm of H-protein,releasing ammonia and producing the reduced H-protein and5,10-methylene-H4folate. The resulting dihydrolipoyl residueof H-protein is reoxidized by L-protein, thereby completing thereaction cycle (7). During the course of the GCS catalytic cycle,H-protein commutes successively among P-, T-, and L-proteinswith its lipoamide arm visiting all three active sites of otherproteins.

Small angle x-ray scattering (8) and cross-linking experi-ments (9) indicated that T-protein and H-protein form a stablecomplex in a 1:1 ratio. NMR spectroscopic studies of the com-plex between H-protein and T-protein indicated that the inter-action surface of H-protein is localized on one side of the cleftwhere the lipoate arm is positioned (6). This work also sug-gested that the role of T-protein is not only to locate H4folate ina position favorable for a nucleophilic attack on the methylenecarbon but also to destabilize the methylamine-loaded H-pro-tein to facilitate unlocking of the arm and to initiate the reac-tion (6). The lack of the N-terminal 16 residues in Escherichiacoli T-protein caused a loss of catalytic activity (10). FurtherN-terminal deletion mutant studies suggested that the N-ter-minal region of T-protein is essential for the conformationalchange of T-protein that accompanies its interaction with H-protein (9). Results of limited proteolysis studies on mutants ofthe E. coli T-protein suggest that the N-terminal region ofT-protein functions as a molecular “hasp” to hold T-protein inthe compact form required for the proper association withH-protein (11).

Until now, three-dimensional structures of two proteins ofthe GCS have been determined, H-protein in four differentforms (apoform, oxidized, methylaminated, and reduced) (12–16) and L-protein (15). Both P-protein and T-protein have beencrystallized (17, 18). In this study, we have determined the firstthree-dimensional structure of T-protein by the multiwave-

* This work was supported by a grant from the Korea Ministry ofScience and Technology (NRL-2001, Grant M1-0104-00-0132). The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

The atomic coordinates and structure factors (codes 1WOS, 1WOO,1WOP, and 1WOR) have been deposited in the Protein Data Bank,Research Collaboratory for Structural Bioinformatics, Rutgers Univer-sity, New Brunswick, NJ (http://www.rcsb.org/).

‡ Both authors contributed equally to this work.§ Recipients of the BK21 fellowship.¶ To whom correspondence should be addressed. Tel.: 82-2-880-6653;

Fax: 82-2-889-1568; E-mail: sewonsuh@snu.ac.kr.1 The abbreviations used are: GCS, glycine cleavage system; NKH,

nonketotic hyperglycinemia; MAD, multiwavelength anomalous dif-fraction; H4folate, tetrahydrofolate; SeMet, selenomethionine; Tm,Thermotoga maritima.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 48, Issue of November 26, pp. 50514–50523, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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length anomalous diffraction (MAD) method. Here we reportthe crystal structure of T-protein from Thermotoga maritima(Tm) in four forms: the apoform; the folinic acid complex; theH4folate complex; and the reduced lipoic acid complex. Thisstudy provides essential structural information on cofactor/inhibitor binding and useful insights into H-protein recognitionby T-protein. It also provides the structural framework forunderstanding the molecular mechanisms of how some mis-sense mutations of the human T-protein lead to its inactivationand NKH (2, 3), a metabolic disorder with severe, frequentlylethal, neurological symptoms in the neonatal period (4).

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The gcvT gene (TM0211) en-coding Tm T-protein was cloned into the expression vector pET-21a(�)(Novagen). The intact protein without any purification tag was overex-pressed in E. coli B834(DE3) cells using terrific broth culture medium.Protein expression was induced by 1 mM isopropyl 1-thio-�-D-galacto-pyranoside, and the cells were incubated for an additional 30 h at 15 °Cfollowing growth to mid-log phase at 37 °C. The cells were lysed bysonication in 50 mM Tris-HCl (pH 7.2) and 200 mM NaCl. Following heattreatment at 80 °C for 10 min, the sample was centrifuged at 18,000rpm for 60 min. The supernatant was applied to a HiLoad 26/10 Q-Sepharose column (Amersham Biosciences), which was previouslyequilibrated with 50 mM Tris-HCl (pH 7.2). Upon eluting with a gradi-ent of NaCl in the same buffer, Tm T-protein was eluted at 250–300 mM

NaCl concentration. The protein was further purified by gel filtrationon a HiLoad XK-16 Superdex 200 prep-grade column (Amersham Bio-sciences), which was previously equilibrated with 50 mM Tris-HCl (pH7.2) and 200 mM NaCl. The procedure for preparing the selenomethi-onine (SeMet)-substituted protein was the same except for the presenceof 10 mM dithiothreitol in all of the buffers used during the purificationsteps. When overexpressing the SeMet-substituted protein in E. coliB834(DE3) cells, we used the M9 cell culture medium that containedextra amino acids including SeMet.

Crystallization—Crystals were grown by the hanging-drop vapordiffusion method at 24 °C by mixing equal volumes (2 �l each) of theprotein solution (27 mg ml�1 concentration in 50 mM Tris-HCl (pH 7.2)and 200 mM NaCl) and the reservoir solution. To grow crystals of thenative protein in the apoform, we used a reservoir solution consisting of15–20% (w/v) polyethylene glycol 3350 and 200 mM sodium dihydrogenphosphate (pH 4.25). The crystals grew to approximate dimensions of0.2 � 0.2 � 0.3 mm within a few days. To grow crystals of the nativeprotein complexed with folinic acid, 1.0 M folinic acid solution (dissolvedin 50 mM Tris-HCl (pH 7.2) and 200 mM NaCl) was mixed with theprotein solution in a 1:20 volume ratio, resulting in an �67-fold molarexcess of folinic acid over the T-protein monomer. The protein mixedwith folinic acid was incubated for 30 min at 4 °C before crystallization.Crystals of the folinic acid complex grew under identical conditions asthe apocrystals. The SeMet-substituted protein was crystallized bymicroseeding techniques under crystallization conditions identical tothose for the native crystals except for the presence of 10 mM dithio-threitol in the protein solution. The crystals of the SeMet-substituted

TABLE IData collection, phasing, and refinement statistics

PLS, Pohang Light Source; BL-6B, Beamline 6B.

Data collection and phasing

Unit cell parameters: a � 52.61 Å, b � 54.16 Å, c � 149.44 Å, � � � � � � 90° (Space group: P212121)X-ray source: PLS BL-6BDataset SeMet � 1 (peak) SeMet � 2 (edge) SeMet � 3 (remote)

Wavelength (Å) 0.97947 0.97935 0.95000Resolution range (Å) 30–2.60 30–2.60 30–2.60Total/unique reflections 232,658/13,749 272,246/13,752 234,700/13,690Completeness (%)a 99.9 (99.9)a 100 (100)a 100 (100)a

Rsym (%)b 12.2 (41.2)b 14.5 (49.2)b 17.8 (78.8)b

Riso (%)c 6.8 (11.5)c 8.0 (16.4)c

f�/f� (e�) �5.5/6.3 �10.4/3.8 �3.0/3.3Figure of meritd for MAD phasing (20–2.80 Å): 0.55/0.72 (before/after density modification)

Refinement

Dataset Apo THF Folinic acid Lipoic acidX-ray source PF (BL-18B) PLS (BL-6B) PLS (BL-6B) PF (BL-5A)X-ray wavelength (Å) 0.98020 1.12714 1.12714 1.00000Space group P212121 P212121 P212121 P212121Unit cell (Å) a � 52.37 a � 52.50 a � 52.57 a � 52.44

b � 53.85 b � 53.94 b � 53.95 b � 53.96c � 149.15 c � 149.40 c � 148.92 c � 149.46

Refinement statisticsResolution range (Å) 20.0–1.84 30–2.40 20–2.00 30–1.95Total/unique reflections 27,2029/36,270 164,708/16,578 407,950/27,459 371,008/30,936Completeness (%) 98.6 98.3 99.0 98.7Rsym (%) 6.0 (15.9)e 6.6 (31.3)e 8.4 (33.7)e 7.2 (21.0)e

Rwork/Rfreef (%) 21.7/24.9 21.7/27.6 21.9/25.8 21.7/24.4

No. of residues (B-factor, Å2) 361 (17.9) 362 (26.6) 362 (21.4) 362 (23.6)No. of waters (B-factor, Å2) 277 (29.2) 124 (32.9) 276 (32.1) 242 (32.6)No. of ligand (B-factor, Å2) 0 1 (27.9) 2 (18.1, 41.8) 1 (62.3)

R.m.s.d. from ideal geometryBonds (Å) 0.0049 0.0077 0.0059 0.0057Angles (°) 1.29 1.32 1.25 1.29

Ramachandran plotMost favorable (%) 92.1 89.0 91.5 92.1Allowed (%) 7.6 11.0 8.5 7.6Generously allowed (%) 0.3 0 0 0.3

a Completeness for I/�(I) � 1.0, high resolution (2.69–2.60 Å) shell in parentheses.b Rsym � hi�I(h)i � I(h)� �/hiI(h)i, where I(h) is the intensity of reflection h, h is the sum over all reflections, and i is the sum over i

measurements of reflection h. Numbers in parentheses reflect statistics for the last shell (2.69–2.60 Å).c Riso � � �FPH� � �FP� �/ � FP�, where FPH and FP are the derivative (�2 or �3) and native (�1) structure factors, respectively. Numbers in

parentheses are for the last shell (2.69–2.60 Å).d Figure of merit � � P(�)ei�/P(�)� �, where � is the phase angle and P(�) is the phase probability distribution.e Numbers in parentheses refer to the last shells (1.91–1.84, 2.49–2.40, 2.09–2.00, and 2.01–1.95 Å, respectively).f R � � �Fobs� � �Fcalc� �/ �Fobs�, where Rfree is calculated for randomly chosen 10% reflections, which were not used for structure refinement, and

Rwork is calculated for the remaining reflections.

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protein grew up to approximate dimensions of 0.2 � 0.2 � 0.3 mmwithin a few days. The crystals of the H4folate complex was obtained bysoaking crystals of the SeMet-substituted protein in a H4folate-satu-rated solution (20% (w/v) polyethylene glycol 3350, 200 mM sodiumdihydrogen phosphate (pH 4.25), and 10 mM dithiothreitol) for 24 hbefore cryoprotection. To obtain the reduced lipoic acid complex, thecrystals of the SeMet-substituted protein were soaked in 50% (v/v)dimethyl sulfoxide solution containing 10 mM dithiothreitol, which waspreviously saturated with lipoic acid, for 2 min before cryoprotection.

X-ray Data Collection and Structure Determination—A crystal of theSeMet-substituted protein was frozen using a cryoprotectant solutioncontaining 25% (v/v) glycerol in the crystallization mother liquor. X-raydiffraction data were collected at 100 K on a Bruker CCD area detectorsystem at the Beamline-6B experimental station of Pohang LightSource. For each image, the crystal was rotated by 1° and the crystal-to-detector distance was set to 360 mm. The raw data were processedand scaled using the program suite HKL2000 (19). The SeMet-substi-tuted crystal belongs to the space group P212121 with unit cell param-eters of a � 52.61 Å, b � 54.16 Å, and c � 149.44 Å. Table I summarizesthe statistics of MAD data collection. All of the eleven expected sele-nium atoms of a monomer in each crystallographic asymmetric unitwere located with the program SOLVE (20), and the selenium siteswere used to calculate the phases with RESOLVE (21). Phasing statis-tics are summarized in Table I. X-ray diffraction data of the H4folatecomplex and folinic acid complex were collected as above. Data of thereduced lipoic acid-bound crystal were collected at 100 K on a Quantum315 CCD detector (Area Detector Systems Corporation, Poway, CA) atthe Beamline-5A experimental station of Photon Factory, whereas thedata of the apoform were collected at the Beamline-18B experimentalstation on an ADSC Quantum 4R CCD detector. The raw data wereprocessed and scaled using the program suite HKL2000 (19).

Model Building and Refinement—Excellent quality of the electrondensity map allowed automatic model building by the program RE-SOLVE (21), giving an initial model that accounted for �70% of thebackbone of the polypeptide chain with much of the sequence assigned.Subsequent manual model building was done using the program O (22).The model was refined with the program CNS (23), including the bulksolvent correction. 10% of the data were randomly set aside as the testdata for the calculation of Rfree (24). Several rounds of model building,simulated annealing, positional refinement, and individual B-factorrefinement were performed. Subsequently, this model was used torefine structures of the apoform, the folinic acid-bound form, theH4folate-bound form, and the lipoic acid-bound form. Refinement sta-tistics are summarized in Table I. All of the models have excellentstereochemistry (Table I) as evaluated by the program PROCHECK(25).

Homology Modeling of Human T-protein—A structural model of thehuman GCS T-protein (Leu33-Phe394) was built by the homology mod-eling server (swissmodel.expasy.org/) using the crystal structure of TmT-protein as template. The N-terminal (Met1-Val32) and C-terminal(Val395-Lys403) regions of the human T-protein were not modeled due toa lack of significant sequence homology.

RESULTS AND DISCUSSION

Overall Structure—We have determined the crystal struc-ture of Tm T-protein by the MAD method and refined fourstructures: (i) the apoform at 1.84 Å; (ii) the complex with(S)-folinic acid (5-formyl-5,6,7,8-tetrahydrofolic acid) at 2.0 Å;(iii) the complex with H4folate at 2.4 Å; and (iv) the complexwith reduced lipoic acid at 1.95 Å. The latter three modelsaccount for residues 1–362 of one T-protein monomer in an

FIG. 1. Overall fold of Tm T-protein.A, ribbon diagram of Tm T-protein. Sec-ondary structure elements were assignedby PROMOTIF (35). Domain 1 (residues1–51 and 140–240), domain 2 (residues52–139 and 241–280), and domain 3 (res-idues 281–362) are colored in purple,green, and cyan, respectively. H4folateand lipoic acid bound near the center ofT-protein are shown in ball-and-stickmodels. This composite figure was pro-duced by incorporating H4folate into thestructure of the lipoic acid complex. Ablue arrow at the bottom indicates thebinding site of the second folinic acid. B,stereo C� trace of Tm T-protein. H4folatewas incorporated into the lipoic acid com-plex to generate this composite structure.Every tenth residue is marked by a blackdot, and every twentieth residue is la-beled. Three signature sequence motifsare highlighted by thick lines: TGYTGE-XGXE motif (residues 186–195) in mag-enta; PXGLGARDXXRhEAXXXLYGmotif (residues 221 and 240) in blue; andGXh(T/S)(S/T)GXXSPTL motif (residues306–317) in green, respectively. The N-terminal region (residues 14–35) of do-main 1, which plays a crucial role in H-protein interaction, is also highlighted byorange thick lines.

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asymmetric unit, whereas Arg362 is additionally missing fromthe apostructure. The missing residues have no electron den-sity. Tm T-protein is monomeric and is oblate-shaped withapproximate dimensions of 55 � 50 � 30 Å (Fig. 1). It istripartite. Its three domains are positioned in a cloverleaf-like arrangement. Domain 1 (residues 1–51 and 140–240)consists of a predominantly antiparallel, six-stranded �-sheetthat contains a single Greek-key motif packed on one side bythree �-helices (�1, �5, and �6) and on the other side by two�-helices (�2 and �7). Domain 2 (residues 52–139 and 241–280), which includes a long excursion from domain 1, has afive-stranded antiparallel �-sheet with flanking �-helices.The two antiparallel �-sheets from domains 1 and 2 areloosely packed against each other. The C-terminal domain 3(residues 281–362) forms a distorted six-stranded jelly rollthat packs perpendicular with the �-sheets of domains 1 and2. The C-terminal tail (residues 354–362) of domain 3 coverspart of domain 1 (Fig. 1, front side) on the opposite side of theN terminus.

DALI structural similarity searches (26) with the Tm T-protein apostructure identified two close relatives: the C-ter-minal H4folate-binding region (residues 421–830) of Ar-throbacter globiformis dimethylglycine oxidase (PDB code1PJ5; a root mean square (r.m.s.) deviation of 1.53 Å for 361equivalent C� positions, a Z-score of 47.3, and a sequenceidentity of 24%) (27) and the E. coli Ygfz protein of unknown

biological function (PDB code 1NRK) (an r.m.s. deviation of 3.2Å for 299 equivalent C� positions, a Z-score of 26.5, and asequence identity of 14%).

Binding Mode of H4folate and Folinic Acid—H4folate-bound and folinic acid-bound structures of Tm T-protein arevirtually identical with an r.m.s. deviation of 0.20 Å for 361C� atoms (Met1-Arg361). H4folate and folinic acid are boundnear the center of Tm T-protein in essentially identical man-ners (Figs. 1 and 2). Both H4folate and folinic acid adopt akinked conformation (Fig. 2). The H4folate-bound structure isalso nearly identical to the apostructure with an r.m.s. devi-ation of 0.23 Å for 361 C� atoms (Met1-Arg361), and thecentral hole has similar solvent accessible pocket volumes inboth the apostructure and the H4folate complex structure.This finding suggests that Tm T-protein has a rigid H4folatebinding pocket. The mouth opening of the central hole is moreopen on the C-terminal side or the glutamate tail side (Fig.1A, front side) than the N-terminal side or the H-proteininteraction side (Fig. 1A, back side). Interactions of T-proteinwith H-protein and the glutamate tail of H4folate are furtherdiscussed below.

Side chains of Asp96, Tyr100, Tyr169, Tyr188, Glu195, Arg227,and Arg362 as well as the carbonyl oxygen of Val110 interactdirectly with H4folate (Fig. 2A). Five of these residues (Asp96,Tyr100, Tyr188, Glu195, and Arg227) are well conserved amongbacterial T-proteins (Fig. 3). Tyr83, Tyr168, Tyr236, Leu237, and

FIG. 2. H4folate binding to the ac-tive site and electron density ofbound ligands. A, stereoview of the ac-tive site around the bound H4folate. Sidechains of the residues lining the activesite are shown together with the mainchain atoms of Val110, Leu237, and Tyr239

that are involved in hydrogen bonding.Black dotted lines denote hydrogen bonds.Blue balls represent water molecules.This figure is drawn with MOLSCRIPT(36) and RASTER3D (37). B, stereoview ofthe active site around the bound folinicacid. C, 2Fo � Fc electron density maps ofthe bound ligands superimposed on folinicacid, H4folate, and lipoic acid (reducedform). Atoms of the ligands are also la-beled. The orientation of H4folate is thesame as in A.

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Tyr239 interact with H4folate indirectly through water mole-cules or through protein main chain atoms. Two of them(Tyr83 and Tyr239) are well conserved among bacterial T-proteins (Fig. 3). The pterin group of H4folate is largelyburied in the central cavity. The side chain O�2 atom of Asp96

makes a hydrogen bond with the N10 nitrogen atom of thepterin group (3.05 Å). The pterin N8 atom interacts with thecarbonyl oxygen of Val110. Both the hydroxyl oxygen atom ofTyr100 and the O�1 atom of Glu195 contact the NA2 atom ofH4folate. The side chain of Arg227 is close to the O4 atomof H4folate. The N2 atom makes a direct hydrogen bond(3.31 Å), whereas the N1 atom is hydrogen-bonded througha water molecule (4.12 Å). The oxygen atom of Tyr169 ishydrogen-bonded to the carbonyl oxygen of the glutamyl

group of H4folate (2.98 Å), whereas the side chain of Arg362

makes a salt bridge with the oxygen O�1 atom of the glutamylgroup of H4folate (3.11 Å). This mode of H4folate binding toTm T-protein resembles that of folinic acid binding to theC-terminal region (residues 430–827) of Arthrobacter globi-formis dimethylglycine oxidase (27). Some other structuralfeatures around H4folate are noteworthy (Fig. 2A). Asn112

and Tyr83 interact with Asp96. Both Asn112 and Tyr83 arehighly conserved in bacteria, whereas only Asn112 is con-served in the human T-protein (as Asn145) and Tyr83 is sub-stituted with Leu116 in human. The separation between theTyr83 O1 atom and the Asp96 O�2 atom is 2.97 Å, whereasthe distance between the Asn112 N�2 atom and the Asp96 O�1atom is 2.79 Å. One water molecule is additionally hydrogen-

FIG. 3. Sequence alignment of bacterial T-proteins. They are Tm (SWISS-PROT accession code Q9WY54); Aa, Aquifex aeolicus (SWISS-PROT accession code O67441); Bs, Bacillus subtilis (SWISS-PROT accession code P54378); Pa, Pseudomonas aeruginosa (SWISS-PROT accessioncode Q9HTX5); and Ec, E. coli (SWISS-PROT accession code P27248). Arrows above the sequences denote �-helices and cylinders �-strands. Bluecircles above the sequence indicate the residues that interact with H4folate with the exception of Asn112, which interacts with folinic acid only.Orange squares below the sequences represent the residues that are close to the bound lipoic acid. Red triangles below the sequences are themissense mutation sites of the human T-protein associated with NKH. Three signature sequence motifs are enclosed by colored boxes: TGYT-GEXGXE motif (residues 186–195) in magenta; PXGLGARDXXRhEAXXXLYG motif (residues 221 and 240) in blue; and GXh(T/S)(S/T)GXXSPTLmotif (residues 306–317) in green, respectively. The N-terminal region (residues 14–35) of domain 1, which plays a crucial role in H-proteininteraction, is also enclosed by a dotted orange box. This figure was drawn with ClustalX (38) and GeneDoc (39).

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bonded to Asp96 (2.89 and 3.17 Å to the O�1 and O�2 atoms ofAsp96, respectively).

In the folinic acid complex, the same residues that contactH4folate interact with folinic acid. Additionally, the oxygenatom of the N5-formyl group of folinic acid interacts with theside chain of Asn112 (Fig. 2B). H4folate lacks a formyl group andthus does not interact with Asn112 (Fig. 2A). The N5-formylgroup is also in proximity of the side chain of Tyr188 with itsaromatic ring nearly perpendicular to the p-aminobenzoic acidgroup (Fig. 2B). In the case of the folinic acid complex, weobserve an additional binding of the second folinic acid at anarrow cleft on the surface of domain 2 near the interfacebetween domains 1 and 2 (Fig. 1A). The second folinic acidtakes an extended conformation, and the residues that interactwith the second folinic acid (the side chains of Glu106, Glu160,Lys173, Ile175, and Glu180; main chain atoms of Met197 andLeu198) are not highly conserved. Furthermore, the averageB-factor of the second folinic acid (41.8 Å2) is much higher thanthe first (18.1 Å2), suggesting a weaker binding of the secondfolinic acid. This secondary binding of folinic acid is probably acrystallization artifact.

It was reported that a pool of polyglutamate forms of folate isdominated by tetraglutamate (25%) and pentaglutamate (55%)in the pea leaf mitochondria (28). The binding affinity ofH4folate polyglutamates for pea leaf T-protein was found to

increase with increasing number of glutamates up to six resi-dues (29). This observation may be explained by the presence ofa surface patch with highly positive electrostatic potential dueto clustering of nine positively charged residues (Lys80, Arg185,Lys280, Lys328, Lys352, Lys353, Arg357, Arg361, and Arg362) in thevicinity of the glutamate tail of H4folate bound to Tm T-protein(Fig. 4A). Five of them (Lys80, Arg185, Lys280, Lys352, andArg357) are well conserved in bacterial T-proteins. The firstglutamate moiety is bound to Tm T-protein in essentially iden-tical manners in both complexes of H4folate and folinic acid(Fig. 2).

Lipoic Acid Binding Reveals Insights into H-protein Recog-nition—To obtain information on the binding site of the lipoylarm of H-protein, we have determined the structure of TmT-protein complexed with the reduced form of lipoic acid. Thelipoic acid-bound structure of Tm T-protein is essentiallyidentical to other forms. The r.m.s. deviations for 361 C�atoms (Met1-Arg361) are 0.13, 0.19, and 0.25 Å for comparingthe lipoic acid-bound structure against the apo, H4folate-bound, and folinic acid-bound structures, respectively. Thisfinding suggests that the lipoic acid-binding site of Tm T-protein is relatively rigid, similar to the H4folate bindingpocket, and the structure of Tm T-protein changes little uponbinding H4folate or lipoic acid. Assuming that the ternarycomplex with H4folate and lipoic acid is structurally similar

FIG. 4. Binding of the glutamate tailof H4folate and lipoic acid and amodel of the complex between T-pro-tein and H-protein. A, positivelycharged residues around the glutamatetail of H4folate. The molecular surface iscolored according to the electrostatic po-tential. The positive electrostatic poten-tial is colored in blue, and the negativepotential is in red. This figure is drawnwith GRASP (40). B, surface diagramshowing the conserved residues aroundthe lipoic acid binding pocket. This view isobtained by an �150° rotation of A. Thecarboxylate group of the bound lipoic acidsticks out of the pocket. The residues thatare strictly conserved in bacterial T-pro-teins are colored in green, and semi-con-served residues are in yellow. An orangecircle denotes the N-terminal portion (res-idues 1–51) of domain 1. C, stereoview ofthe T-protein active site. H4folate hasbeen incorporated into the structure ofthe lipoic acid complex. Two sulfur atomsof lipoic acid are labeled. Side chains ofthe residues lining the active site areshown. Black dotted lines depict hydrogenbonds. Blue balls represent water mole-cules. D, a model of the complex betweenT-protein and H-protein. T-protein is rep-resented by the electrostatic potential atthe molecular surface. The backbone ofT. thermophilus H-protein (Protein DataBank code 1ONL) is drawn in blue tubeswith a green arrow indicating the proba-ble direction of Lys63 to which the lipoylmoiety is attached. A half-transparentred dot near Lys289 (corresponding toE. coli Lys288) of Tm T-protein indicatesthe location of Glu42 (corresponding to E.coli Asp43) of T. thermophilus H-protein.This figure is drawn with GRASP (40).

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to the binary complexes with H4folate, folinic acid, or lipoicacid, we incorporated H4folate of the H4folate complex intothe model of the lipoic acid complex to facilitate discussion(Figs. 1 and 4C).

Lipoic acid is bound in an �15-Å deep pocket adjacent to theH4folate binding pocket with its carboxylate group pointingtoward the bulk solvent (Figs. 1 and 4C). The S6 sulfur atommakes a hydrogen bond with the side chain of Asp228 at adistance of 2.90 Å. Arg227 (N2 atom) is close to the S8 sulfuratom (3.23 Å). Phe20, Tyr188, Leu224, and Leu238 as well as thealiphatic part of the Arg227 side chain surround the aliphaticpart of lipoic acid (Fig. 4C). Leu224, Arg227, Asp228, and Leu238

are all strictly conserved in bacterial T-proteins, and theybelong to the highly conserved sequence motif PXGLGAR-DXXRhEAXXXLYG between positions 221 and 240 in domain1 of Tm T-protein (boxed in blue in Fig. 3; highlighted in thickblue lines in Fig. 1B), where X stands for any amino acid andh is a hydrophobic residue. This motif is the longest signaturesequence of T-protein (H4folate-dependent aminomethyl-transferase). Tyr188 belongs to another conserved sequencemotif TGYTGEXGXE between positions 186 and 195 in do-main 1 of Tm T-protein (boxed in magenta in Fig. 3; high-lighted in thick magenta lines in Fig. 1B). This motif also

contains the strictly conserved residue Glu195, which directlycontacts H4folate (Fig. 1B). Bacterial T-proteins have a thirdconserved sequence motif GXh(T/S)(S/T)GXXSPTL betweenpositions 306 and 317 in domain 3 of Tm T-protein (boxed ingreen in Fig. 3; highlighted in thick green lines in Fig. 1B),where two possible residues are grouped within parentheses.This signature motif in domain 3 is not directly involved incatalysis or the binding of H4folate and lipoic acid. It appearsto be important for H-protein recognition (further discussedbelow).

Two structurally conserved water molecules are bound be-tween lipoic acid and H4folate (Wat47 and Wat114 in Fig. 4C).They are present in all four structures except in the folinic acidcomplex where Wat114 is absent, because the N5-formyl groupoccupies the site of Wat114. Wat114 is hydrogen-bonded to theS8 atom of lipoic acid at a distance of 2.83 Å and to the O�1atom of Asn112 at 2.88 Å. Wat47 is hydrogen-bonded to thebackbone nitrogen atom of Tyr239 at a distance of 2.74 Å and tothe O�1 atom of Asp96 at 2.84 Å. If a methylamine group werecovalently attached to the S8 atom of lipoic acid, Asn112 (O�1oxygen) and Tyr188 (O1 oxygen) would be within hydrogen-bonding distances from the methylamine group. Asn112 andTyr188 are strictly conserved in T-proteins (Fig. 3).

FIG. 5. Sequence alignment of the human T-protein and close-up views of some mutation sites associated with NKH. A, sequencealignment of the human T-protein against Tm T-protein. Red triangles below the sequences are the missense mutation sites of the human T-proteinassociated with NKH. Light green triangles above the sequences are the residues that interact with the mutated residues in the human T-proteinmodel. Colored boxes are the same as in Fig. 3. B, stereoview of the residues around His10 in Tm T-protein. Side chains of Ala15, Pro26, Tyr29, andAsp47 are shown. C, stereoview of the residues around Asp242 in Tm T-protein. Side chains of Arg231, Leu281, Arg309, and Ser310 are shown. D,stereoview of the residues around Asn112 in Tm T-protein. Side chains of Tyr83, Asp96, Tyr188, and Leu237 are shown as well as the main chain atomsof Leu237 and folinic acid. A red ball represents a water molecule.

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The binding site of lipoic acid clearly suggests the possibleinterface for interaction with H-protein. The carboxylate groupof lipoic acid bound in the pocket points toward the solvent onthe N-terminal side with the other end pointing toward thepterin group of H4folate (Figs. 1B and 4B). There is a deep cleftbetween domains 1 and 3 on this side of Tm T-protein (Fig. 4D).Along this cleft around lipoic acid, many conserved residues areclustered, including Phe20, Leu224, Arg227, Asp228, Leu238, andTyr239 (Fig. 4B). This cleft appears to be the site of H-proteinbinding. Deletions of the N-terminal 4, 7, 11, and 16 residuesfrom the E. coli T-protein led to reduction in the activity to 42,9, 4, and 0%, respectively, relative to the wild-type enzyme (9,10). Our Tm T-protein structure revealed that the N-terminalregion of domain 1 (circled in Fig. 4B) contributes to one sideof this cleft and that removal of the N-terminal residues 1–13would seriously affect the folding of the sequence region14–35 (boxed in orange in Fig. 3; highlighted in thick orangelines in Fig. 1B), causing the distortion of the H-proteinbinding surface. Therefore, the loss of E. coli T-protein activ-ity caused by a deletion of the N-terminal 16 residues is dueto disruption of the H-protein-binding site. It is also apparentthat the signature sequence motif GXh(T/S)(S/T)GXXSPTL indomain 3 (boxed in green in Fig. 3; highlighted in thick greenlines in Fig. 1B) provides another side of the H-protein-binding cleft (Fig. 1B).

Cross-linking experiments indicated that Lys288 of E. coliT-protein is close to Asp43 of E. coli H-protein when they forma 1:1 complex (9). E. coli Lys288 corresponds to Tm Lys289 andis conserved as lysine or arginine in bacterial T-proteins (Fig.3). The location of Lys289 in Tm T-protein is shown in Fig. 4, Band D. The lipoyl moiety is covalently attached to Lys63 (indi-cated by a green arrow in Fig. 4D) of T. thermophilus H-protein(16), which has a 57% sequence identity with Tm H-protein. Allof these pieces of information allowed us to build a crude butreasonable model of the complex between T-protein and H-

protein (Fig. 4D). In this model, H-protein is positioned alongthe cleft between domains 1 and 3 of T-protein on the N-terminal side, Glu42 (corresponding to E. coli Asp43) of T. ther-mophilus H-protein (marked by a half-transparent red dot inFig. 4D) is close to Lys289 of Tm T-protein (corresponding toE. coli Lys288), and Lys63 of T. thermophilus H-protein pointstoward the lipoic acid binding pocket (as indicated by a greenarrow in Fig. 4D). It was suggested that H-protein undergoes asmall conformational change upon binding to T-protein so thatthe lipoyl arm carrying the methylamine group is exposed (6).Upon interaction with H-protein, T-protein may also undergo asmall structural alteration such as limited domain rearrange-ment. To characterize the possible structural changes in bothT-protein and H-protein that accompany the complex forma-tion, further structural studies are required.

Structural Understanding of Nonketotic Hyperglycin-emia—A considerable level of sequence similarity exists be-tween the human T-protein and Tm T-protein (Fig. 5A): 33%identity between the human T-protein (Leu33-Phe394) and TmT-protein (Met1-Phe355). Thus, the homology-modeled struc-ture of the human T-protein is highly similar to that of TmT-protein in its core. Only the surface regions (Ser68-His71,Gly171-Ala178, Gly217-Val220, His242-Ile247, Leu292-Ala299, andGln311-Arg315 of human) that are not directly associated withthe catalytic machinery show significant structuraldeviations.

A number of mutations in the human T-protein gene havebeen identified among NKH patients, including missense mu-tations that lead to the following amino acid substitutions:G269D, G47R, and R320H (30); D276H (31); H42R (32); E211K,C95V (4), and N145I (33); and R296H, V212A, and Y225C (34).The spatial locations of these mutations are shown in Fig. 6A.All of these residues with the exception of Arg296 do not belongto the above-listed variable surface regions. The correspond-ence of the human T-protein mutation sites to Tm T-protein is

FIG. 6. Missense mutation sites ofthe human T-protein in NKH. A, stereoC� trace of the homology model of thehuman T-protein. Side chains of theeleven mutated residues in NKH aredrawn in red. B, stereoview of the residuesaround Asp276, equivalent of Tm Asp242,in the human T-protein model. Sidechains of Arg265, Arg320, Thr348, andSer349 are shown. This is the same view asFig. 5C.

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as follows: His42 (human)/His10 (Tm); Gly47 (human)/Ala15

(Tm); Cys95 (human)/Val62 (Tm); Asn145 (human)/Asn112 (Tm);Glu211 (human)/Ile175 (Tm); Val212 (human)/Val176 (Tm); Tyr225

(human)/Tyr188 (Tm); Gly269 (human)/Thr235 (Tm); Asp276 (hu-man)/Asp242 (Tm); Arg296 (human)/Lys262 (Tm); and Arg320

(human)/Leu281 (Tm). Cys95 (human)/Val62 (Tm), Glu211 (hu-man)/Ile175 (Tm), Val212 (human)/Val176 (Tm), and Arg296 (hu-man)/Lys262 (Tm) belong to the highly variable sequence re-gions. Other residues are more conserved and seem to beimportant in the formation of the H4folate binding pocket andin providing the interface for H-protein recognition. Besides themissense mutations, a premature stop codon Q192X (4), aone-base deletion 183delC (31), and a splice site mutationIVS7-1G � A (4) were also reported. They would result inhighly altered polypeptides, thus causing the loss in the activ-ity of the human T-protein.

Among the missense mutation sites, Asn145 (human)/Asn112

(Tm) and Tyr225 (human)/Tyr188 (Tm) are strictly conservedamong both bacterial and human T-proteins (Figs. 3 and 5A).Asp276 (human)/Asp242 (Tm) is conserved as either Asp or Glu(Figs. 3 and 5A). Arg320 (human)/Leu281 (Tm) is strictly con-served in bacterial T-proteins only (Fig. 3). Gly269 (human)/Thr235 (Tm) is variable in the sequence but is within the sig-nature sequence motif PXGLGARDXXRhEAXXXLYG indomain 1, whereas Asp276 (human)/Asp242 (Tm) is right afterthis motif (Fig. 3). Tyr225 (human)/Tyr188 (Tm) belongs to thesignature sequence motif TGYTGEXGXE in domain 1 (Fig. 3).The side chains of both Asn112 (Tm) and Tyr188 (Tm) are closeto the oxygen atom of the N5-formyl group of folinic acid (Fig.5D) as mentioned above. Thus, the mutations of their equiva-lents in the human T-protein would cause a considerable loss inthe T-protein activity. Mutation of Asn145 (human)/Asn112 (Tm)into histidine will affect H4folate binding both directly andindirectly by disturbing the network involving Asp96, whichinteracts with the N10 atom of H4folate (Fig. 5D). Mutation ofTyr225 (human)/Tyr188 (Tm) into cysteine will seriously alterthe THF binding pocket.

His42 (human)/His10 (Tm) and Gly47 (human)/Ala15 (Tm) aresemi-conserved among both bacterial and human T-proteins(Figs. 3 and 5A). His10 (Tm), Ala15 (Tm), and Asp242 (Tm) areslightly separated from either the H4folate binding pocket orthe lipoic acid-binding site (Fig. 1B). Mutations at these threesites would have no direct effect on the binding of H4folate orlipoic acid, but they appear to alter the H-protein bindinginterface. Interestingly, the residues interacting with His10

(Tm) and Asp242 (Tm) (Ala15, Pro26, Tyr29, and Asp47; Arg231,Thr309, and Ser310) are well conserved in the T-protein family(Fig. 3). Details of the interactions around His10 (Tm) andAsp242 (Tm) are shown in Fig. 5, B and C, respectively. In TmT-protein, Asp47 and Tyr29 stabilize the imidazole ring of His10

by hydrogen bonding and the side chain of Pro26 lies close to theimidazole ring of His10. The side chain of Ala15 points towardthe hydrogen-bonding network around His10 (Fig. 5B). Thus,the H42R and G47R mutations in the human T-protein couldpossibly disrupt the network involving these residues, thusaltering the proper conformation of the N-terminal portion ofdomain 1, which provides the interface for interaction withH-protein as discussed above. In Tm T-protein, Arg231, Thr309,and Ser310 are clustered around Asp242, forming a hydrogen-bonding network and a salt bridge (Fig. 5C). Arg231 is part ofthe signature motif PXGLGARDXXRhEAXXXLYG in domain1, whereas Thr309 and Ser310 belong to the GXh(T/S)(S/T)GXX-SPTL motif in domain 3 (Fig. 3). Mutation of Asp276 (human)/Asp242 (Tm) into histidine could possibly perturb the hydrogen-bonding network around this residue and might alter thesurface features that are crucial for H-protein binding.

Val176 (Tm), corresponding to Val212 (human), makes a hy-drophobic core with neighboring residues (Leu155, Val159,Val183, and Leu196 of Tm), of which Leu155, Val159, and Val183

are conserved in human, whereas Leu196 is replaced with Ile233

in human (Fig. 5A). Thus, it is expected that the mutation ofVal212 (human)/Val176 (Tm) into alanine would destabilize thishydrophobic core. Leu281 (Tm), corresponding to Arg320 (hu-man), is conserved among bacterial T-proteins only. It is notpart of the H4folate- or lipoic acid-binding site but is close to theconserved Asp242 (Tm). The environment around Asp242 (Tm) isshown in Fig. 5C. In the human T-protein model, Arg320 makesan additional salt bridge with Asp276 (Fig. 6B). This interactionmay be necessary for the proper function or stability of thehuman T-protein, thus explaining why the R320H mutationcauses the impaired T-protein activity.

Acknowledgments—We thank Dr. H. S. Lee and staff at BeamlineBL-6B of Pohang Light Source for assistance during data collection. Wealso thank Prof. N. Sakabe and staff for assistance during data collec-tion at Photon Factory, Beamlines BL-5A and BL-18B.

REFERENCES

1. Douce, R., Bourguignon, J., Neuburger, M., and Rebeille, F. (2001) TrendsPlant Sci. 6, 167–176

2. Tada, K., and Kure, S. (1993) J. Inherit. Metab. Dis. 16, 691–7033. Sakata, Y., Owada, Y., Sato, K., Kojima, K., Hisanaga, K., Shinka, T., Suzuki,

Y., Aoki, Y., Satoh, J., Kondo, H., Matsubara, Y., and Kure, S. (2001) BrainRes. Mol. Brain Res. 94, 119–130

4. Toone, J. R., Applegarth, D. A., Coulter-Mackie, M. B., and James, E. R. (2000)Mol. Genet. Metab. 70, 116–121

5. Toone, J. R., Applegarth, D. A., Coulter-Mackie, M. B., and James, E. R. (2001)Hum. Mutat. 17, 76–76

6. Guilhaudis, L., Simorre, J. P., Blackledge, M., Marion, D., Gans, P.,Neuburger, M., and Douce, R. (2000) Biochemistry 39, 4259–4266

7. Motokawa, Y., Fujiwara, K., and Okamura-Ikeda, K. (1995) in Bithiols inHealth and Disease (Packer, L., and Cadenas, E., eds) pp. 389–407, MarcelDekker, Inc., New York

8. Okamura-Ikeda, K., Ohmura, Y., Fujiwara, K, and Motokawa, Y. (1993) Eur.J. Biochem. 216, 539–548

9. Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1999) Eur. J. Biochem.264, 446–452

10. Okamura-Ikeda, K., Kameoka, N., Fujiwara, K., and Motokawa, Y. (2003)J. Biol. Chem. 278, 10067–10072

11. Cohen-Addad, C., Faure, M., Neuburger, M., Ober, R., Sieker, L.,Bourguignon, J., Macherel, D., and Douce, R. (1997) Biochimie (Paris) 79,637–643

12. Pares, S., Cohen-Addad, C., Sieker, L., Neuburger, M., and Douce, R. (1994)Proc. Natl. Acad. Sci. U. S. A. 91, 4850–4853

13. Macherel, D., Bourguignon, J., Forest, E., Faure, M., Cohen-Addad, C., andDouce, R. (1996) Eur. J. Biochem. 236, 27–33

14. Cohen-Addad, C., Pares, S., Sieker, L., Neuburger, M., and Douce, R. (1995)Nature Struct. Biol. 2, 63–68

15. Faure, M., Bourguignon, J., Neuburger, M., Macherel, D., Sieker, L., Ober, R.,Kahn, R., Cohen-Addad, C., and Douce, R. (2000) Eur. J. Biochem. 267,2890–2898

16. Nakai, T., Nakagawa, N., Maoka, N., Masui, R., Kuramitsu, S., and Kamiya,N. (2003) Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 554–557

17. Nakai, T., Ishijima, J., Masui, R., Kuramitsu, S., and Kamiya, N. (2003) ActaCrystallogr. Sect. D Biol. Crystallogr. 59, 1610–1618

18. Lokanath, N. K., Kuroishi, C., Okazaki, N., and Kunishima, N. (2004) ActaCrystallogr. Sect. D Biol. Crystallogr. 60, 1450–1452

19. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–32620. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol.

Crystallogr. 55, 849–86121. Terwilliger, T. C. (2003) Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 38–4422. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crys-

tallogr. Sect. A 47, 110–11923. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-

Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read,R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr.Sect. D Biol. Crystallogr. 54, 905–921

24. Brunger, A. T. (1992) Nature 355, 472–47425. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993)

J. Appl. Crystallogr. 26, 283–29126. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123–13827. Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038–404828. Besson, V., Rebeille, F., Neuburger, M., Douce, R., and Cossins, E. A. (1993)

Biochem. J. 292, 425–43029. Rebeille, F., Neuburger, M., and Douce, R. (1994) Biochem. J. 302, 223–22830. Nanao, K., Okamura-Ikeda, K., Motokawa, Y., Danks, D. M., Baumgartner,

E. R., Takada, G., and Hayasaka, K. (1994) Hum. Genet. 93, 655–65831. Kure, S., Shinka, T., Sakata, Y., Osamu, N., Takayanagi, M., Tada, K.,

Matsubara, Y., and Narisawa, K. (1998) J. Hum. Genet. 43, 135–13732. Kure, S., Mandel, H., Rolland, M. O., Sakata, Y., Shinka, T., Drugan, A.,

Boneh, A., Tada, K., Matsubara, Y., and Narisawa, K. (1998) Hum. Genet.102, 430–434

Structure of Glycine Cleavage System T-protein50522

by guest on July 15, 2020http://w

ww

.jbc.org/D

ownloaded from

33. Toone, J. R., Applegarth, D. A., Coulter-Mackie, M. B., and James, E. R. (2001)Mol. Genet. Metab. 72, 322–325

34. Toone, J. R., Applegarth, D. A., Levy, H. L., Coulter-Mackie, M. B., and Lee, G.(2003) Mol. Genet. Metab. 79, 272–280

35. Hutchinson, E. G., and Thornton, J. M. (1996) Protein Sci. 5, 212–22036. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946–950

37. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505–52438. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins,

D. G. (1997) Nucleic Acids Res. 25, 4876–488239. Nicholas, K. B., Nicholas, H. B., Jr., and Deerfield D. W., II (1997) Embnet.

News 4, 1440. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281–296

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Hyung Ho Lee, Do Jin Kim, Hyung Jun Ahn, Jun Yong Ha and Se Won SuhBASIS FOR UNDERSTANDING NONKETOTIC HYPERGLYCINEMIA

BINDING, INSIGHTS INTO H-PROTEIN RECOGNITION, AND MOLECULAR Crystal Structure of T-protein of the Glycine Cleavage System: COFACTOR

doi: 10.1074/jbc.M409672200 originally published online September 7, 20042004, 279:50514-50523.J. Biol. Chem. 

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