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, 94 (18), 9550-5

Substrate-induced Conformational Change in a Trimeric Ornithine Transcarbamoylase

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Substrate-induced Conformational Change in a Trimeric Ornithine Transcarbamoylase

Y Ha et al. Proc Natl Acad Sci U S A.

Abstract

The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.

Figures

Figure 5
Figure 5
Domain closure. The Cα trace of the loop region and side chains of relevant residues are shown. (Upper Left) Unligated P. aeruginosa catabolic OTCase structure, with the active site open to solvent (13). (Lower Left) E. coli OTCase/PALO structure, with the two domains closed. (Right) The simulated annealing omit Fo − Fc map (contour level 2.0 σ, residues 233–239, and PALO omitted from minimization and phase calculation), confirms the positions of these residues and the ligand bound.
Figure 1
Figure 1
Superposition of E. coli OTCase onto P. aeruginosa catabolic OTCase and E. coli ATCase catalytic subunit, generated with the 78 Cα coordinates of the 5 β-strands and 4 α-helices of the CP binding domain as defined in E. coli ATCase (35). The rmsd between structures are 0.53 Å and 0.91 Å, respectively. CP, CP binding domain; l-orn, l-ornithine binding domain. (Upper) Superposition of E. coli OTCase ligated with PALO (thick line) on unligated P. aeruginosa catabolic OTCase (thin line), with Cα of Gly-237 labeled for comparison. (An ∗ is used in unligated P. aeruginosa catabolic OTCase.) (Lower) Superposition of E. coli OTCase ligated with PALO (thick line) on E. coli ATCase ligated with PALA (thin line).
Figure 2
Figure 2
Sequence and secondary structure alignment of E. coli OTCase (ecootc), P. aeruginosa catabolic OTCase (paeotc), and E. coli ATCase catalytic subunit (ecoatc). Residues corresponding to α-helices and β-strands are separated by a space from the rest of the sequence and underlined with dashed and solid lines, respectively (13, 35). Residues at the N/C termini and the beginning and end of secondary structural elements in E. coli OTCase are numbered.
Figure 3
Figure 3
Cartoon drawings of E. coli OTCase catalytic trimer ligated with the bisubstrate analog PALO, shown as a space filling model. These figures were generated by graphic programs molscript (57) and render (58, 59). Chain A1, A2, and A3 are colored light blue, blue, and green respectively. (Upper) Top view, down the molecular three-fold axis. CP and l-ornithine binding domains of chain A1 are labeled. One active site, shared between chain A1 and the 80s loop of chain A2, is also labeled. (Lower) Side view, perpendicular to the three-fold axis. N/C termini and α helices 1, 7, 8a, and 9a of chain A2 are labeled.
Figure 4
Figure 4
Stereoview showing the interaction of the bisubstrate analog PALO with active site residues and interactions of Arg-57 and PALO with Gln-82, Lys-86, and Glu-87 of the 80s loop from an adjacent polypeptide chain. The interactions between NZ of Lys-86 with OE1 of Gln-82 and OT1 of PALO appear to be bridged by disordered water molecules.

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