Structure of unligated aspartate carbamoyltransferase of Escherichia coli at 2.6-A resolution.

Abstract

The three-dimensional structure of the allosteric enzyme aspartate carbamoyltransferase (EC 2.1.3.2) has been refined to a crystallographic R-factor of 0.24 at 2.6-A resolution in the space group P321, where a and b are 122.1 A and c is 142.2 A. This structure is isomorphous to the form of the enzyme complexed to the allosteric inhibitor cytidine tri-phosphate. All sources of sequence information have been evaluated against the electron density. The corrected amino acid sequences of the catalytic and regulatory proteins have been incorporated in the model, and three regions in the active site are described: (i) near arginine-105, histidine-134, and arginine-167, (it) near lysine-232 and arginine-229, and (iii) near lysine-83 and lysine-84.The allosteric enzyme aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli catalyzes the formation of carbamoyl-L-aspartate from L-aspartate and carbamoyl phosphate. The enzyme consists of catalytic trimers C3 and regulatory dimers r2 in a structure c6r6 having D3 symmetry (1-3). Substrates induce very large conformational changes in the structure of the enzyme (4) which are influenced by the allosteric inhibitor CTP and the allosteric activator ATP (5-7).Three-dimensional structures have been reported (8) for the T (less active) form of the enzyme in the unligated form (space group R32) and in the CTP-ligated form (space group P321). We report here the structure to a resolution of 2.6 A of the unligated T form in the space group P321, isomorphous with the CTP form. The improved structure provides two new crystallographic environments for the unligated rc unit. In addition, we have rebuilt the model with the use of a primary structure, which is based on DNA sequences, in both the c and r chains (2, 9-11). Although no change occurs in the trace of either the c or the r chain, the insertion of residues after 220 of the c chain (9, 10) has required the relocation of amino acid side chains from 217 to 261. In addition, the insertion of a lysine at 129 increases the number of residues in the r chain from 152 (2) to 153 (11) and requires a small revision in the three-dimensional structure.MATERIALS AND METHODS Aspartate carbamoyltransferase of E. coli was purified by the method of Gerhart and Holoubek (12), as described elsewhere (8). Single crystals of the enzyme were grown at room temperature by dialysis of a solution containing 5-10 mg of protein per ml against a solution containing 100 mM maleic acid, 10 mM Tris HCI, 1 mM 2-mercaptoethanol, 0.2 mM EDTA, and 0.3 mM NaN3 at pH 5.8. After 3-7 days, platelike crystals formed with typical dimensions of 1.2 x 1.0 x 0.2 mm. These crystals are hexagonal, in the space group P321, and had unit cell dimensions of a = b = 122.1 A and c = 142.2 A.X-ray diffraction data were collected by the oscillation method, using an Elliott GX6 rotating anode unit operated at about 39 kV and 19 mA, and using a 100-,um focusing cup. Each oscillation photograph was taken over a 10 inte...

Significance of structural changes in proteins: Expected errors in refined protein structures

“…Eliminating true structural differences in extracting errors There are real differences between multiple molecules in the asymmetric unit due to differences in the packing environment "The four-letter code refers to the PDB designation for each structure, for which the references are: lTHB (Waller & Liddington, 1990); 2CCY (Finzel et al, 1985); 4CHA (Tsukada & Blow, 1985); 4DFR (Bolin et al, 1982); ZHHB (Fermi et al, 1984); 3CYT (Takano & Dickerson, 1980); ZAZA (Baker, 1988); l G D l (Skarzynski et al, 1987); lAZA (Norris et al, 1983);lGPl (Epp et al, 1983); 1HMQ (Stenkamp et al, 1982);2pKA (Bodeetal., 1983); 2PFK (Rypniewski Evans, 1989); 1FCB (Xia & Mathews, 1990); 4MDH (Birktoft et al, 1989); 4ATc (Ke et al, 1984); ~F C I (Deisenhofer, 1981); IHBS (Padlan & Love, 1985). of the independent molecules.…”

Mechanism of Inactivation of Ornithine Transcarbamoylase by N δ-(N′-Sulfodiaminophosphinyl)-l-ornithine, a True Transition State Analogue?

“…The atomic coordinates and structure factors (code 1DUV) of these structures with unliganded enzyme structures (17,18) demonstrate that OTCase and ATCase undergo a conformational change upon substrate binding, bringing substrates together and facilitating condensation (14,19). Specifically, in OTCases, the binding of substrates and/or inhibitors induces a conformational change in the 240s loop 2 to form a closed conformation.…”

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

“…Two functional and structural states of the enzyme have been characterized by a variety of techniques: a less active, low-affinity, T-state, stabilized by CTP and a more active, high-affinity, R-state stabilized by the substrates or the bisubstrate analog Nphosphonacetyl-L-aspartate (PALA). The corresponding crystal structures (11,12) recently refined (13,14) described the T-to-R transition as an 11-Å (1 Å ϭ 0.1 nm) expansion along the 3-fold axis together with subunit and domain rotations (15) resulting in disruption of the r1-c4 interface between regulatory and catalytic chains. This quaternary rearrangement is accompanied by tertiary changes in both the catalytic and regulatory chains, most notably the 80s and 240s loops of the catalytic chains.…”