Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: implications for allosteric regulation

Abstract

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-A resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.

Figure 1

Structure of the C trimer–PALA complex at 1.95-Å resolution. (A) Ribbon representation of the PALA-liganded C trimer. Each chain is shown in a different color with the N-terminal, carbamoyl-phosphate-binding domain (center), depicted in a lighter shade than the C-terminal, aspartate-binding, domain. PALA is shown in blue Corey–Pauling–Koltun rendering, highlighting both the interchain and interdomain location of the active sites. (B) 2F_o − F_c electron density map (contour level 1σ) in the region of the bound bisubstrate analog. (C) Omit F_o − F_c map of the inhibitor, PALA (contour level 3σ), using phases calculated from a model excluding the PALA molecule.

Figure 2

The catalytic chains in the C trimer exhibit pronounced hinge closure upon PALA binding and adopt a conformation very similar to that of the chains in the PALA-bound holoenzyme. (A) Superposition of the N-terminal domains of catalytic chains from the C trimer–PALA complex (red) and the unliganded C trimer (PDB ID 3csu, Y chain, green). The chains superimpose closely within each domain but exhibit a 9.5° difference in hinge angle. (B) Superposition of catalytic chains from the C trimer–PALA complex (red) and the holoenzyme–PALA complex (PDB ID 1D09, blue). The similarity of the liganded conformations indicates that PALA binding is sufficient to promote the closed hinge in the holoenzyme. PALA is shown in red as a space-filling model. (C) Shift plots depicting differences in C^α positions as a function of residue number. Shift corresponding to A (chain from the C trimer–PALA complex versus the Y chain of the unliganded C trimer) is shown as a thin line illustrating a large relative motion of one domain with a hinge near Leu-140. Shift plot corresponding to B (chain from the C trimer–PALA complex versus a catalytic chain from the holoenzyme–PALA complex) is shown as a thick line. This comparison shows close similarity, except at the termini and the 240s loop.

Figure 3

Similar active-site conformations in PALA-liganded C trimer and holoenzyme. (A) Comparison of the three active sites in the C trimer–PALA complex, based on the superposition of all backbone atoms, shows nearly identical conformations. (B) Comparison of an active site in the C trimer–PALA complex (red) with that in the holoenzyme–PALA complex (blue). The conformation of PALA is very similar in both structures and is shown for only the C trimer–PALA complex. Slight differences are observed in rotamers of Arg-54.