2.5 Å structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-l-aspartate☆

In an X-ray diffraction study using the method of multiple isomorphous replacement, the structure of aspartate Carbamoyltransferase (EC 2.1.3.2) complexed with the bisubstrate analog N-(phosphonacetyl)-l-aspartate (PALA)‡ has been solved to 2.5 A. Ten rounds of model building and 123 cycles of restrained reciprocal space refinement have resulted in a model containing 94.4% of the theoretical atoms of the protein-inhibitor complex with an R-factor of 0.231. The fit of the model to the density is excellent, except for occasional side-chains and two sections of the regulatory chains that may be disordered. The electron density for the PALA molecule is readily identifiable for both catalytic (c) chains of the asymmetric unit and bonding interactions with several important residues including Ser52, Arg54, Thr55, Ser80, Lys84, Arg105, His134, Arg165, Arg229 and Gln231 are apparent. The carboxylate groups of the PALA molecule are in a nearly cis conformation. Gross quaternary changes between the T and R forms are noted and in agreement with earlier work from this laboratory. Namely, in the new structure the catalytic trimers move apart by 12 A along the 3-fold axis of the enzyme and relocate by 10 ° relative to each other, adopting a more eclipsed position. The regulatory (r) chains in the new structure reorient about their 2-fold axis by 15 °. Large tertiary changes that include domain migration and rearrangement are also present between these two forms. In the R form both domains of the catalytic chain relocate closer to each other in order to bind to the inhibitor. The polar domain seems to bind primarily to the carbamoyl phosphate moiety of PALA, and the equatorial domain binds primarily to the l-aspartate moiety. Other changes in tertiary structure bring the 80s loop (from an adjacent catalytic chain) and the 240s loop into a position to interact with the PALA molecule. Changes have been searched for in all interface regions of the enzyme. While the C1–C4§ and C1–R4 regions have been completely altered, most of the other interchain interfaces are similar in the T and R forms. The intrachain interfaces, between domains of the same catalytic chains, have undergone some reorganization as these domains move closer to each other when the inhibitor is bound. This new structure allows a reinterpretation of genetic and chemical modification studies done to date. As such, the roles of Ser52, Lys84 and His134 are supported. Other residues implicated in similar studies such as Cys47, Tyr165, Lys232 and Tyr240 are too far from the inhibitor to have a direct interaction. Finally, on the basis of the above results, a preliminary model for the homotropic transition is offered. In it, the change to the R-form is triggered by the binding of l-aspartate to the equatorial domain of a catalytic chain in which carbamoyl phosphate is bound to its site in the polar domain. Each C1–C4 group within the enzyme appears to be able to adopt its more active form somewhat independently, but the reorientation of the regulatory dimers that accompanies the homotropic transition serves to facilitate a T to R state transition in the other C1–C4 units. During the change from the T to R states most contact areas between rc pairs and between rr pairs are preserved. This is consistent with the suggestion that c-r-r-c is a functionally important unit. In addition, direct communication between C1–C4 is also consistent with the new structure.