The structure description and discussion will be mainly based on this subunit.Oligomerization State in SolutionTo determine the states of oligomerization of both complete NAGS and the NAT domain in solution, cross-linking and analytic gel filtration experiments were performed. Cross-linkingFigure 1. Biochemical properties of hNAT. A and B, dependence of enzyme activity on the concentration of AcCoA or L-glutamate. AcCoA or Lglutamate was varied in the range of 0.25?.0 and 0.50?0.0, respectively, with L-glutamate and AcCoA fixed at 10 or 2.5 mM, respectively. doi:10.1371/journal.pone.0070369.gStructure of Human N-Acetyl-L-Glutamate SynthaseFigure 2. Oligomeric structure of mNAGS and hNAGS in solution. A: Analytic gel chromatography of mNAGS and hNAGS. Elution profiles of mNAGS and hNAGS are shown in dashed and solid lines, respectively. B: Analytic gel chromatography of mNAT and hNAT. Elution profiles of mNAT and hNAT 16574785 are shown in dashed and solid lines, respectively. C: Cross-linking of mNAGS. Lanes 1; protein size markers; 2, mNAGS (2.5 mg) without cross-linking reagent; 3, mNAGS (2.5 mg) with cross-linking reagent, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt; 4, mNAGS with cross-linking reagent, dimethyl suberimidate dihydrochloride. D: Cross-linking of mNAGS. Lanes 1; protein size markers; 2, mNAGS without crosslinking reagent; 3, mNAGS (1.5 mg) with cross-linking reagent, dimethyl suberimidate dihydrochloride; 4, mNAGS (4.5 mg) with cross-linking reagent, dimethyl suberimidate dihydrochloride. doi:10.1371/journal.pone.0070369.gEach subunit has a central 1315463 seven-strand b-sheet arranged as a Vshaped structure with three anti-parallel b-strands in the Cterminal arm and four anti-parallel b-strands in the N-terminal arm (Figure 3A). The central b-sheet is flanked by five helices with three helices on one side and four helices on the other. The structure has a typical fold of GCN5-related N-acetyltransferase and is similar to the NAT domain structure of the bifunctional NAGS/K from M. maris (Table 2).DimerizationEven though four subunits were identified in an asymmetric unit, the PISA server [10] indicated that the stable molecule isdimer. Subunit A and subunit B form a molecular dimer. The molecular dimers for subunit X and subunit Y were generated via crystallographic two-fold symmetries, respectively. At each dimer interface (A-B, X-X or Y-Y), the C-terminal arm from one subunit interacts with the C-terminal arm from the other subunit to form a continuous 6-strand antiparallel b-sheet, similar to the NAT-NAT domain interaction observed in the MedChemExpress BTZ-043 mmNAGS/K structure (Figure 3C). This extensive interface has a buried interface of ?1477 A2. The interactions in this interface Licochalcone A involve extensive mainchain (Asp490, Ser492 and Ser494) and side-chain (Ser494) hydrogen bonding interactions, p-p interactions (Phe496?Structure of Human N-Acetyl-L-Glutamate SynthaseTable 1. Data collection and refinement statistics.?Table 2. RMSD values (A) among different subunit within NAG bound structure and with the bifunctional mmNAGS/K native structure.Data collection Bound ligands Space group ?Wavelength (A) ?Resolution (A) ?Unit-cell parameters (A) NAG P43212 1.0 50?.10 (2.14?.10) a = b = 116.1 c = 109.7 Measurements Unique reflections Redundancy Completeness ( ) ,I/s(I). Rmerg( )b Refinement ?Resolution range (A) No. of protein atoms No. of water atoms No. of hetero atoms ?Rmsd of bond lengths (A) Rmsd of bond angle (u) Rwork ( )c Rfree (.The structure description and discussion will be mainly based on this subunit.Oligomerization State in SolutionTo determine the states of oligomerization of both complete NAGS and the NAT domain in solution, cross-linking and analytic gel filtration experiments were performed. Cross-linkingFigure 1. Biochemical properties of hNAT. A and B, dependence of enzyme activity on the concentration of AcCoA or L-glutamate. AcCoA or Lglutamate was varied in the range of 0.25?.0 and 0.50?0.0, respectively, with L-glutamate and AcCoA fixed at 10 or 2.5 mM, respectively. doi:10.1371/journal.pone.0070369.gStructure of Human N-Acetyl-L-Glutamate SynthaseFigure 2. Oligomeric structure of mNAGS and hNAGS in solution. A: Analytic gel chromatography of mNAGS and hNAGS. Elution profiles of mNAGS and hNAGS are shown in dashed and solid lines, respectively. B: Analytic gel chromatography of mNAT and hNAT. Elution profiles of mNAT and hNAT 16574785 are shown in dashed and solid lines, respectively. C: Cross-linking of mNAGS. Lanes 1; protein size markers; 2, mNAGS (2.5 mg) without cross-linking reagent; 3, mNAGS (2.5 mg) with cross-linking reagent, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt; 4, mNAGS with cross-linking reagent, dimethyl suberimidate dihydrochloride. D: Cross-linking of mNAGS. Lanes 1; protein size markers; 2, mNAGS without crosslinking reagent; 3, mNAGS (1.5 mg) with cross-linking reagent, dimethyl suberimidate dihydrochloride; 4, mNAGS (4.5 mg) with cross-linking reagent, dimethyl suberimidate dihydrochloride. doi:10.1371/journal.pone.0070369.gEach subunit has a central 1315463 seven-strand b-sheet arranged as a Vshaped structure with three anti-parallel b-strands in the Cterminal arm and four anti-parallel b-strands in the N-terminal arm (Figure 3A). The central b-sheet is flanked by five helices with three helices on one side and four helices on the other. The structure has a typical fold of GCN5-related N-acetyltransferase and is similar to the NAT domain structure of the bifunctional NAGS/K from M. maris (Table 2).DimerizationEven though four subunits were identified in an asymmetric unit, the PISA server [10] indicated that the stable molecule isdimer. Subunit A and subunit B form a molecular dimer. The molecular dimers for subunit X and subunit Y were generated via crystallographic two-fold symmetries, respectively. At each dimer interface (A-B, X-X or Y-Y), the C-terminal arm from one subunit interacts with the C-terminal arm from the other subunit to form a continuous 6-strand antiparallel b-sheet, similar to the NAT-NAT domain interaction observed in the mmNAGS/K structure (Figure 3C). This extensive interface has a buried interface of ?1477 A2. The interactions in this interface involve extensive mainchain (Asp490, Ser492 and Ser494) and side-chain (Ser494) hydrogen bonding interactions, p-p interactions (Phe496?Structure of Human N-Acetyl-L-Glutamate SynthaseTable 1. Data collection and refinement statistics.?Table 2. RMSD values (A) among different subunit within NAG bound structure and with the bifunctional mmNAGS/K native structure.Data collection Bound ligands Space group ?Wavelength (A) ?Resolution (A) ?Unit-cell parameters (A) NAG P43212 1.0 50?.10 (2.14?.10) a = b = 116.1 c = 109.7 Measurements Unique reflections Redundancy Completeness ( ) ,I/s(I). Rmerg( )b Refinement ?Resolution range (A) No. of protein atoms No. of water atoms No. of hetero atoms ?Rmsd of bond lengths (A) Rmsd of bond angle (u) Rwork ( )c Rfree (.
