Refined Crystal Structures of Unligated Adenylosuccinate Synthetase from Escherichia coli

Abstract

Crystal structures of unligated adenylosuccinate synthetase from Escherichia coliin space groups P2 1and P2 12 12 1have been refined to R-factors of 0.199 and 0.206 against data to 2.0 and 2.5 Å, respectively. Bond lengths and angles deviate from expected values by 0.011 Å and 1.7° for the P2 1crystal form and by 0.015 Å and 1.7° for the P2 12 12 1crystal form. The fold of the polypeptide chain is dominated by a central β-sheet, which is composed of nine parallel strands and a tenth antiparallel strand. Extending off from this central β-sheet are four subdomains. The four subdomains contribute loops of residues that are disordered or have high thermal parameters. At least three of these loops (residues 42 to 52, 120 to 131 and 298 to 304) contribute essential residues to the putative active site of the synthetase. In the absence of ligands, much of the active site of the synthetase exists in an ill-defined conformational state. Two, nearly independent regions contribute residues to the interface between polypeptide chains of the synthetase dimer. A pair of helices (H4 and H5) interact with their symmetry-equivalent mates by way of residues that are not conserved amongst the known sequences of the synthetase. The second interface region involves conserved residues belonging to structural elements that connect strands of the central β-sheet. Residues putatively involved in the binding of IMP lie at or near the interface between polypeptide chains of the dimer. Of the four sequence elements putatively common to all GTP hydrolases, the synthetase has only the guanine recognition element and a glycine-rich loop (P-loop). Although the base recognition element is essentially identical with those of the p21 rasand G αproteins, the P-loop of the synthetase is extended in size relative to the P-loops of other GTP hydrolases. The P-loop has two acid residues (Asp13 and Glu14), which are found in the P-loops of only the synthetase family. Glu14 may be involved in the stabilization of the enlarged P-loop of the synthetase, whereas Asp13 may play a role in catalysis and in the coordination of Mg 2+. The structural elements of the p21 rasand G αproteins responsible for binding Mg 2+are either absent from the synthetase or unavailable for the coordination of metal cations.