Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit

Abstract

The lack of knowledge of the three-dimensional structure of the trimeric, catalytic (C) subunit of aspartate transcarbamoylase (ATCase) has impeded understanding of the allosteric regulation of this enzyme and left unresolved the mechanism by which the active, unregulated C trimers are inactivated on incorporation into the unliganded (taut or T state) holoenzyme. Surprisingly, the isolated C trimer, based on the 1.9-A crystal structure reported here, resembles more closely the trimers in the T state enzyme than in the holoenzyme:bisubstrate-analog complex, which has been considered as the active, relaxed (R) state enzyme. Unlike the C trimer in either the T state or bisubstrate-analog-bound holoenzyme, the isolated C trimer lacks 3-fold symmetry, and the active sites are partially disordered. The flexibility of the C trimer, contrasted to the highly constrained T state ATCase, suggests that regulation of the holoenzyme involves modulating the potential for conformational changes essential for catalysis. Large differences in structure between the active C trimer and the holoenzyme:bisubstrate-analog complex call into question the view that this complex represents the activated R state of ATCase.

Figure 1

Structure of the ATCase C trimer. (A) The quality of the electron density maps is illustrated by the 20- to 1.88-Å resolution, 2Fo-Fc map contoured at 1σ, showing residues of the phosphate-binding loop, Ser-52 through Thr-55. (B) Ribbon diagram (21) of the X (red), Y (green), and Z (gold) chains of the C trimer, viewed along the trimerization axis. The active sites, which contain residues from adjacent chains, are designated by asterisks.

Figure 2

Structural differences among the catalytic chains in the C trimer. (A) Ribbon representation of the catalytic chains superimposed (22) by using the carboxyl-terminal domains (residues 150–229 and 252–284) of chains X (red), Y (green), and Z (gold). An asterisk marks the active site. Differences in the interdomain hinge angle in the three chains are apparent from the displacement of the nonsuperimposed, amino-terminal domains. The superimposed, carboxyl-terminal domains show close correspondence. Two flexible loops (80s and 240s) containing active-site residues differ among the chains and exhibit disordered regions, shown as breaks in the ribbons. (B) Shifts in Cα positions of chains X and Z superimposed by the backbone atoms of the amino-terminal (residues 1–73 and 90–134; thick, solid line) or carboxyl-terminal (thin, dashed line) domain, plotted as a function of residue number. Gaps result from disordered regions in one or both chains. The individual domains of the C trimer closely resemble each other (<0.3-Å shifts) in tertiary structure, except for the loop regions. Large shifts in the nonsuperimposed domains reflect the hinge-angle variation.

Figure 3

The C trimer is globally more similar to the T state holoenzyme. (A) Differences in interdomain hinge angles in the catalytic chains of the isolated C trimer, the T state, and the PALA complex of the holoenzyme. The differences of up to 4.8° in hinge angle of the independent X, Y, and Z chains illustrate the asymmetry of the isolated C trimer. In contrast, the C trimers in the holoenzyme structures show exact 3-fold symmetry, and the upper and lower trimers differ in hinge angle by only 0.9° for the T state enzyme and 0.2° for the PALA complex (8, 9). The chains of the isolated C trimer are 1.3–6.0° more open than the catalytic chains of the T state holoenzyme and 8.4–12.3° more open than those in the PALA-liganded holoenzyme structure. (B) Ribbon diagram of chain Z (gold) and catalytic chains from the T state (blue) and PALA-complexed holoenzyme (magenta) superimposed by using the Cα atoms of the carboxyl-terminal domain. An asterisk marks the active site. Interdomain hinge angles are similar for chain Z and the catalytic chain in the T state holoenzyme, and the hinge is more closed in the PALA complex. (C) Shifts in Cα positions between the Z chain of the isolated C trimer and the T state (blue) and PALA complex (magenta) of the holoenzyme. The chains were superimposed by using the amino-terminal (thick lines) or carboxy-terminal (thin lines) domains. With the exception of the 240s loop, which makes a contact between trimers in the crystal, the main chain of the C trimer is more similar to the T state holoenzyme.

Figure 4

Similar local conformations near the active sites of the isolated C trimer and the T state holoenzyme. Residues are labeled in the one-letter code. (A) Catalytic (dark blue) and regulatory (r, light blue) chains from the T state holoenzyme superimposed by the amino-terminal domain of the catalytic chains on chain Z (gold) of the C trimer. The 80s loop on the right is flexible in the isolated trimer, and the 240s loop on the left is ordered in chain Z by a crystal contact. (B) Superposition of the active site regions of chain Z (gold), a catalytic chain from the T state (blue), and PALA-liganded (magenta) holoenzyme structures. For clarity, PALA was omitted from the liganded structure. The ordered regions of the active sites in the isolated C trimer more closely resemble the T state holoenzyme. Diagnostic similarities are apparent for Thr-168 and the phosphate binding loop (Ser-52, Arg-54, and Thr-55). Because the isolated C trimer defines an active conformation, the distinct tertiary structure of the holoenzyme:PALA complex may result largely from ligand binding rather than the allosteric transition alone.