Crystal structure of recombinant human T-cell cyclophilin A at 2.5 A resolution.

The structure of the unligated human T-cell recombinant cyclophilin has been determined at 3 A resolution by multipole isomorphous replacement methods and refined at 2.5 A resolution to an R factor of 0.209. The root-mean-square errors of the bond lengths and bond angles are 0.013 A and 2.8 degrees from ideal geometry, respectively. The overall structure is a beta-barrel, consisting of eight antiparallel beta-strands wrapping around the barrel surface and two alpha-helices sitting on the top and the bottom closing the barrel. Inside the barrel, seven aromatic and other hydrophobic residues form a compact hydrophobic core. A loop of Lys-118 to His-126 and four beta-strands (B3-B6) constitute a pocket we speculate to be the binding site of cyclosporin A.

Conformational transition of fructose-1,6-bisphosphatase: structure comparison between the AMP complex (T form) and the fructose 6-phosphate complex (R form).

A structure of the neutral form of fructose-1,6-bisphosphatase complexed with AMP has been determined by the molecular replacement method and refined at a 2.5-A resolution to a crystallographic R factor of 0.169. The root-mean-square errors of the structure from standard geometry are 0.013 A for bond lengths and 2.99 degrees for bond angles. Comparison of the AMP complex with the F6P complex shows that dimer C3-C4 twists about 19 degrees about a molecular 2-fold axis when dimers C1-C2 of the R and T forms of the enzyme are superimposed one another and that a slight shift of about 1 A of the AMP domain partially compensates this twist. The R to T transition of the enzyme does not significantly change the conformation of the F6P-binding site. However, residues at the divalent metal site and the AMP site show significant positional shifts. If these results can be extended to substrate in place of F6P, they suggest that regulation of the enzyme by AMP may occur partly through effects on metal-ion affinity or position. AMP binds to the same sites of the T and R forms, but only half-occupancy was observed in the alkaline R form. Sequential binding of AMP, at least in pairs, is suggested as the unligated R form is converted to the T form. Two possible pathways are suggested for allosteric communication over about 28 A between the AMP site and the active site: one via helices H1, H2, and H3 and another via the eight-stranded beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of the neutral form of fructose-1,6-bisphosphatase complexed with the product fructose 6-phosphate at 2.1-A resolution.

The crystal structure of fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with the product fructose 6-phosphate (F6P) has been refined at 2.1-A resolution to an R factor of 0.177 with root-mean-square deviations of 0.014 A and 2.9 degrees from the ideal geometries of bond lengths and bond angles, respectively. The secondary structures but not the trace of the unligated enzyme have been slightly revised in the F6P complex at this higher resolution. Helix H4 in the unligated structure has been refined to a helix-like coil, and two very short 3(10) helices have been found, one in H4 and one in H5. F6P at 10 mM concentration in the absence of divalent metals in our study shows major binding at the active site and minor binding at the AMP site. The major site has almost equal full occupancy in the C1 and C2 chains of the crystallographic asymmetric unit, while the minor site shows occupancy only in the C1 chain at about 50%. The electron density in both (2Fo - Fc) and (Fo - Fc) maps calculated by omitting F6P slightly favors the beta anomer of D-F6P over the alpha anomer. Possible functions of the active-site residues are discussed, and candidates are suggested for site-directed mutagenesis.

Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 6-phosphate, AMP, and magnesium.

The crystal structure of fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with fructose 6-phosphate, AMP, and Mg2+ has been solved by the molecular replacement method and refined at 2.5-A resolution to a R factor of 0.215, with root-mean-square deviations of 0.013 A and 3.5 degrees for bond lengths and bond angles, respectively. No solvent molecules have been included in the refinement. This structure shows large quaternary and tertiary conformational changes from the structures of the unligated enzyme or its fructose 2,6-bisphosphate complex, but the secondary structures remain essentially the same. Dimer C3-C4 of the enzyme-fructose 6-phosphate-AMP-Mg2+ complex twists about 19 degrees relative to the same dimer of the enzyme-fructose 2,6-bisphosphate complex if their C1-C2 dimers are superimposed on one another. Nevertheless, many interfacial interactions between dimers of C1-C2 and C3-C4 are conserved after quaternary structure changes occur. Residues of the AMP domain (residues 6-200) show large migrations of C alpha atoms relative to barely significant positional changes of the FBP domain (residues 201-335).

Structure refinement of fructose-1,6-bisphosphatase and its fructose 2,6-bisphosphate complex at 2.8 A resolution.

The structures of the native fructose-1,6-bisphosphatase (Fru-1,6-Pase), from pig kidney cortex, and its fructose 2,6-bisphosphate (Fru-2,6-P2) complexes have been refined to 2.8 A resolution to R-factors of 0.194 and 0.188, respectively. The root-mean-square deviations from the standard geometry are 0.021 A and 0.016 A for the bond length, and 4.4 degrees and 3.8 degrees for the bond angle. Four sites for Fru-2,6-P2 binding per tetramer have been identified by difference Fourier techniques. The Fru-2,6-P2 site has the shape of an oval cave about 10 A deep, and with other dimensions about 18 A by 12 A. The two Fru-2,6-P2 binding caves of the dimer in the crystallographically asymmetric unit sit next to one another and open in opposite directions. These two binding sites mutually exchange their Arg243 side-chains, indicating the potential for communication between the two sites. The beta, D-fructose 2,6-bisphosphate has been built into the density and refined well. The oxygen atoms of the 6-phosphate group of Fru-2,6-P2 interact with Arg243 from the adjacent monomer and the residues of Lys274, Asn212, Tyr264, Tyr215 and Tyr244 in the same monomer. The sugar ring primarily contacts with the backbone atoms from Gly246 to Met248, as well as the side-chain atoms, Asp121, Glu280 and Lys274. The 2-phosphate group interacts with the side-chain atoms of Ser124 and Lys274. A negatively charged pocket near the 2-phosphate group includes Asp118, Asp121 and Glu280, as well as Glu97 and Glu98. The 2-phosphate group showed a disordered binding perhaps because of the disturbance from the negatively charged pocket. In addition, Asn125 and Lys269 are located within a 5 A radius of Fru-2,6-P2. We argue that Fru-2,6-P2 binds to the active site of the enzyme on the basis of the following observations: (1) the structure similarity between Fru-2,6-P2 and the substrate; (2) sequence conservation of the residues directly interacting with Fru-2,6-P2 or located at the negatively charged pocket; (3) a divalent metal site next to the 2-phosphate group of Fru-2,6-P2; and (4) identification of some active site residues in our structure, e.g. tyrosine and Lys274, consistent with the results of the ultraviolet spectra and the chemical modification. The structures are described in detail including interactions of interchain surfaces, and the chemically modifiable residues are discussed on the basis of the refined structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structure of the Glu-239----Gln mutant of aspartate carbamoyltransferase at 3.1-A resolution: an intermediate quaternary structure.

The structure of the unligated Glu 239----Gln mutant of Escherichia coli aspartate carbamoyltransferase (EC 2.1.3.2) has been determined to 3.1-A resolution and refined to a crystallographic residual of 0.22 in the space group P321. The unit-cell dimensions of the unligated enzyme are a = 122.3 A, c = 147.1 A. The c axis cell length is intermediate between the c axis lengths of the T (tense)(c = 142.2 A) and R (relaxed) (c = 156.2 A) state structures. Furthermore, the quaternary structure of the mutant enzyme is intermediate between the quaternary structures of the T form and the R form. The differences between the quaternary structures of the Glu-239----Gln and T-form enzymes can be described as follows: the separation between the catalytic trimers increases by approximately 1.5 A along the threefold axis, and they each rotate in opposite directions approximately 0.5 degree around the threefold axis, whereas the regulatory dimers rotate approximately 2 degrees around the twofold axes.

Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms.

The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry. The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155). We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively). The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain. The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations. The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees. No changes occur in secondary structure. Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit. However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme. Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains. The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism. The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding. On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o

Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli.

The protein and solvent structure of the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli yields an R-factor of 0.155 for data to a resolution of 2.6 A. The model has 7353 protein atoms, 945 sites for solvent, and two molecules of CTP. A total of 25 of the 912 residues of the model exist in more than one conformation. The root-mean-square deviation of bond lengths and angles from their ideal values is 0.013 A and 2.1 degrees, respectively. The model reported here reflects a correction in the trace of the regulatory chain. One molecule of CTP binds to each of the two regulatory chains of the asymmetric unit of the crystal. The interactions between the pyrimidine of each CTP molecule and the protein are similar. The 4-amino group of CTP binds to the carbonyl groups of residues 89 (tyrosine) and 12 (isoleucine) of the regulatory chain. The nitrogen of position 3 of the pyrimidine binds to the amide group of residue 12; the 2-keto group binds to lysine 60. The 2'-OH group of the ribose forms hydrogen bonds with lysine 60 and the carbonyl group of residue 9 (valine). The binding of the phosphate groups of CTP to the regulatory chain probably reflects an incomplete association of CTP with the enzyme at pH 5.8. A lattice contact influences the interaction between the triphosphate group of one CTP molecule and the protein. For the other CTP molecule, only lysine 94 binds to the phosphate groups of CTP. Of the two regulatory and two catalytic chains of the asymmetric unit of the crystal, there are only two significant violations of non-crystallographic symmetry. The active site in the vicinity of arginine 54 of one catalytic chain is larger than the active site of its non-crystallographic mate. The "expanded" cavity accommodates four solvent molecules in the vicinity of arginine 54 as opposed to two molecules of water for the "contracted" cavity. Furthermore, arginine 54 in the "expanded" pocket adopts two conformations, either hydrogen-bonding to glutamate 86 or to the phenolic oxygen atom of tyrosine 98; residues 86 and 98 are in a catalytic chain related by 3-fold symmetry to the catalytic chain of arginine 54. In the "contracted" pocket, arginine 54 binds only to glutamate 86.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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 triphosphate. 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, (ii) near lysine-232 and arginine-229, and (iii) near lysine-83 and lysine-84.

Gross quaternary changes in aspartate carbamoyltransferase are induced by the binding of N-(phosphonacetyl)-L-aspartate: A 3.5-A resolution study.

The three-dimensional structure of the complex of N-(phosphonacetyl)-L-aspartate with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) has been determined to a nominal resolution of 3.5 A by single-crystal x-ray diffraction methods. Initial phases were obtained by the method of "molecular tectonics": beginning with the structure of the CTP-protein complex, the domains of the catalytic and regulatory chains were manipulated as separate rigid bodies. The resulting coordinates were used to calculate an electron density map, which was then back transformed to give a set of calculated amplitudes and phases. Using all observed data, we obtained a crystallographic R factor between observed and calculated amplitudes Fo and Fc of 0.46. An envelope was then applied to a 2Fo - Fc map and the density was averaged across the molecular twofold axis. Two cycles of averaging yielded an R factor of 0.25. In this complex, we find that the two catalytic trimers have separated from each other along the threefold axis by 11-12 A and have rotated in opposing directions around the threefold axis such that the total relative reorientation is 8-9 degrees. This rotation places the trimers in a more nearly eclipsed configuration. In addition, two domains in a single catalytic chain have changed slightly their spatial relationship to each other. Finally, the two chains of one regulatory dimer have rotated 14-15 degrees around the twofold axis, and the Zn domains have separated from each other by 4 A along the threefold axis. These movements enlarge the central cavity of the molecule and allow increased accessibility to this cavity through the six channels from the exterior surface of the enzyme.