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The crystal structure of Escherichia coli aspartate carbamoyltransferase
complexed with adenosine 5'-triphosphate (ATP) has been solved by molecular
replacement and has been refined to a crystallographic residual of 0.17 at 2.6-A
resolution by using the computer program X-PLOR. The unit cell dimensions of
this crystal form are a = b = 122.2 A and c = 143.3 A and the space group is
P321. Although the c-axis unit cell dimension is approximately 1 A longer than
the corresponding dimension of the CTP-ligated P321 crystal form (c = 142.2 A),
the ATP-ligated enzyme adopts a T-like quaternary structure. The base moiety of
ATP interacts with residues Glu10, Ile12, and Lys60 while the ribose is near
Asp19 and Lys60; the triphosphate entity is bound to Lys94, although His20 and
Arg96 are nearby. We observe a higher occupancy for ATP in the allosteric site
of the R1 regulatory chain in comparison to the occupancy of the R6 allosteric
site. These crystallographically independent sites are related by a molecular
2-fold axis. There are other violations of the noncrystallographic symmetry that
are similar to those observed in the refined CTP-ligated aspartate
carbamoyltransferase structure. These infringements on the molecular symmetry
might be the result of intermolecular interactions in the crystal. To ensure the
most meaningful comparison with the ATP-ligated structure, we refined the
previously reported CTP-bound and unligated structures to crystallographic
residuals between 0.17 and 0.18 using X-PLOR. These X-PLOR refined structures
are not significantly different from the initial structures that had been
crystallographically refined by a restrained least-squares method. After making
all possible comparisons between the CTP- and ATP-ligated and the unligated
T-state structures, we find that the most significant differences are located at
the allosteric sites and in small changes in the quaternary structures. At the
allosteric site, the binding of CTP and ATP successively enlarges the nucleotide
binding cavity, particularly in the vicinity of the base. The changes in the
quaternary structure can be characterized by an increase in the separation of
the catalytic trimers by approximately 0.5 A as ATP binds to the unligated T
structure. On the basis of these structural studies, we discuss the
relationships between the conformational differences in the allosteric site and
the small changes in the quaternary structure within the T form to the possible
mechanisms for CTP inhibition and ATP activation.
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