Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli

Atomic models representing the electron density of two crystalline forms of aspartate carbamoyltransferase from Escherichia coli are reported here. The unliganded form (R32 crystal symmetry) and the CTP-liganded form (P321 crystal symmetry) have been refined independently at resolutions of 3.0 å and 2.8 Å, respectively, each to a crystallographic R-factor of 27%. The molecular models include at least 95% of the theoretical number of atoms for the aspartate Carbamoyltransferase molecule based on chemical sequence information. We provide details of the refinement process for the two structures, and an evaluation of the accuracy of the molecular models.For the most part, the regulatory and catalytic chains of the unliganded enzyme and the CTP-liganded form are in similar conformations. Large conformational differences in the CTP and native forms exist, however, specifically in the region of CTP binding to the regulatory chain. In addition, a segment of ten amino acid residues, which includes Lys83 and Lys84 of the catalytic chain, is disordered in the CTP-liganded form, in contrast to the native structure, where the same residues have refined well into density.Each catalytic monomer of aspartate carbamoyltransferase is in contact with three catalytic chains and two regulatory monomers. Each regulatory monomer borders on one other regulatory chain and two catalytic chains. The catalytic trimera are in contact in the hexamer; residues important to homotropic effects and catalysis (Tyr165 and Tyr232) are integral parts of the interface. We present a thorough survey of interface regions, cataloging polar interactions between sidechains throughout the molecule.We discuss, in context with the present structures, the chemical modifications and mutations of the enzyme. Highlighted specifically are Cys47, Tyr165 and Tyr232, Lys83, Lys84, Trp209 and Trp279 and Gly128, residues of demonstrated importance to the catalytic of regulatory function or aspartate carbamoyltransferase. The spatial arrangement of “active site” residues argues for a catalytic pocket shared between two monomers within catalytic subunit.